Method of identifying negative hormone and/or antagonist activities

ABSTRACT

Aryl-substituted and aryl and (3-oxo-1-propenly)-substituted benzopyran, benzothiopyran, 1,2-dihydroquinoline, and 5,6-dihydronaphthalene derivatives have retinoid negative hormone and/or antagonist-like biological activities. The invented RAR antagonists can be administered to mammals, including humans, for the purpose of preventing or diminishing action of RAR agonists on the bound receptor sites. Specifically, the RAR agonists are administered or coadministered with retinoid drugs to prevent or ameliorate toxicity or side effects caused by retinoids or vitamin A or vitamin A precursors. The retinoid negative hormones can be used to potentiate the activities of other retinoids and nuclear receptor agonists. For example, the retinoid negative hormone called AGN 193109 effectively increased the effectiveness of other retinoids and steroid hormones in in vitro transactivation assays. Additionally, transactivation assays can be used to identify compounds having negative hormone activity. These assays are based on the ability of negative hormones to down-regulate the activity of chimeric retinoid receptors engineered to possess a constitutive transcription activator domain.

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)of the three following U.S. applications, each of which was filed as anonprovisional application and converted to a provisional application byseparate petitions filed on Jan. 31, 1996: Application No. 08/522,778,filed Sep. 1, 1995; application No. 08/522,779, filed Sep. 1, 1995; andapplication No. 08/542,648, filed Oct. 13, 1995. The completedisclosures of these related applications is hereby incorporated hereinby this reference thereto.

FIELD OF THE INVENTION

The present invention relates to novel compounds having retinoidnegative hormone and/or retinoid antagonist-like biological activities.More specifically, the invention relates to 4-aryl substitutedbenzopyran, 4-aryl substituted benzothiopyran, 4-aryl substituted1,2-dihydroquinoline and 8-aryl substituted 5,6-dihydronaphthalenederivatives which may also be substituted by a substituted3-oxo-1-propenyl group. These novel compounds have retinoid antagonistlike-activity and are useful for treating or preventing retinoid andvitamin A and vitamin A precursor induced toxicity in mammals and as anadjunct to treatment of mammals with retinoids to prevent or ameliorateunwanted or undesired side effects. The invention further relates to theuse of retinoid negative hormones for increasing the biologicalactivities of other retinoids and steroid hormones and inhibiting thebasal activity of unliganded retinoic acid receptors.

BACKGROUND OF THE INVENTION

Compounds which have retinoid-like activity are well known in the art,and are described in numerous United States and other patents and inscientific publications. It is generally known and accepted in the artthat retinoid-like activity is useful for treating mammals, includinghumans, in order to cure or alleviate the symptoms associated withnumerous diseases and conditions.

Retinoids (vitamin A and its derivatives) are known to have broadactivities, including effects on cell proliferation and differentiation,in a variety of biological systems. This activity has made retinoidsuseful in the treatment of a variety of diseases, includingdermatological disorders and cancers. The prior art has developed alarge number of chemical compounds which have retinoid-like biologicalactivity, and voluminous patent and chemical literature existsdescribing such compounds. The relevant patent literature includes U.S.Pat. Nos. 4,980,369, 5,006,550, 5,015,658, 5,045,551, 5,089,509,5,134,159, 5,162,546, 5,234,926, 5,248,777, 5,264,578, 5,272,156,5,278,318, 5,324,744, 5,346,895, 5,346,915, 5,348,972, 5,348,975,5,380,877, 5,399,561, 5,407,937, (assigned to the same assignee as thepresent application) and patents and publications cited therein, whichparticularly describe or relate to chroman, thiochroman and1,2,3,4-tetrahydroquinoline derivatives which have retinoid-likebiological activity. In addition, several applications are pending whichare assigned to the assignee of the present application, and which aredirected to further compounds having retinoid-like activity.

U.S. Pat. Nos. 4,740,519 (Shroot et al.), 4,826,969 (Maignan et al.),4,326,055 (Loeliger et al.), 5,130,335 (Chandraratna et al.), 5,037,825(Klaus et al.), 5,231,113 (Chandraratna et al.), 5,324,840(Chandraratna), 5,344,959 (Chandraratna), 5,130,335 (Chandraratna etal.), Published European Patent Application Nos. 0 176 034 A (Wuest etal.), 0 350 846 A (Klaus et al.), 0 176 032 A (Frickel et al.), 0 176033 A (Frickel et al.), 0 253 302 A (Klaus et al.), 0 303 915 A (Bryceet al.), UK Patent Application GB 2190378 A (Klaus et al.), GermanPatent Application Nos. DE 3715955 Al (Klaus et al.), DE 3602473 Al(Wuest et al., and the articles J. Amer. Acad. Derm. 15: 756-764 (1986)(Sporn et al.), Chem. Pharm. Bull. 33: 404-407 (1985) (Shudo et al.), JMed Chem. 31: 2182-2192 (1988) (Kagechika et al.), Chemistry and Biologyof Synthetic Retinoids CRC Press Inc. 1990 pp. 334-335, 354 (Dawson etal.), describe or relate to compounds which include a tetrahydronaphthylmoiety and have retinoid-like or related biological activity. U.S. Pat.No. 4,391,731 (Boller et al.) describes tetrahydronaphthalenederivatives which are useful in liquid crystal compositions.

An article by Kagechika et al. in J Med. Chem 32: 834 (1989) describecertain6-(3-oxo-1-propenyl)-1,2,3,4-tetramethyl-1,2,3,4-tetrahydronaphthalenederivatives and related flavone compounds having retinoid-like activity.The articles by Shudo et al. in Chem. Pharm. Bull 33: 404 (1985) and byJetten et al. in Cancer Research 47: 3523 (1987) describe or relate tofurther 3-oxo-1-propenyl derivatives (chalcone compounds) and theirretinoid-like or related biological activity.

Unfortunately, compounds having retinoid-like activity (retinoids) alsocause a number of undesired side effects at therapeutic dose levels,including headache, teratogenesis, mucocutaneous toxicity,musculoskeletal toxicity, dyslipidemias, skin irritation, headache andhepatotoxicity. These side effects limit the acceptability and utilityof retinoids for treating disease.

It is now general knowledge in the art that two main types of retinoidreceptors exist in mammals (and other organisms). The two main types orfamilies of receptors are respectively designated as the RARs and RXRs.Within each type there are subtypes: in the RAR family the subtypes aredesignated RAR-α, RAR-β and RAR-γ, in RXR the subtypes are: RXR-α, RXB-βand RXR-γ. Both families of receptors are transcription factors that canbe distinguished from each other based on their ligand bindingspecificities. All-trans-RA (ATRA) binds and activates a class ofretinoic acid receptors (RARS) that includes RAR-α, RAR-β and RAR-γ. Adifferent ligand, 9-cis-RA (9C-RA), binds and activates both the RARsand members of the retinoid X receptor (RXR) family.

It has also been established in the art that the distribution of the twomain retinoid receptor types, and of the several subtypes is not uniformin the various tissues and organs of mammalian organisms. Moreover, itis generally accepted in the art that many unwanted side effects ofretinoids are mediated by one or more of the RAR receptor subtypes.Accordingly, among compounds having agonist-like activity at retinoidreceptors, specificity or selectivity for one of the main types orfamilies, and even specificity or selectivity for one or more subtypeswithin a family of receptors, is considered a desirable pharmacologicalproperty.

Relatively recently compounds have been developed in the art which bindto RAR receptors without triggering the response or responses that aretriggered by agonists of the same receptors. The compounds or agentswhich bind to RAR receptors without triggering a "retinoid" response arethus capable of blocking (to lesser or greater extent) the activity ofRAR agonists in biological assays and systems. More particularly,regarding the scientific and patent literature in this field, publishedPCT Application WO 94/14777 describes certain heterocyclic carboxylicacid derivatives which bind to RAR retinoid receptors and are said inthe application to be useful for treatment of certain diseases orconditions, such as acne, psoriasis, rheumatoid arthritis and viralinfections. A similar disclosure is made in the article by Yoshimura etal. J Med. Chem. 38: 3163-3173 (1995). Kaneko et al. Med. Chem Res. 1:220-225 (1991); Apfel et al. Proc. Natl. Acad. Sci. USA 89: 7129-7133Augusty 1992 Cell Biology; Eckhardt et al. Toxicology Letters 70:299-308 (1994); Keidel et al. Molecular and Cellular Biology 14: 287-298(1994); and Eyrolles et al. J. Med. Chem. 37: 1508-1517 (1994) describecompounds which have antagonist like activity at one or more of the RARretinoid subtypes.

In addition to undesirable side-effects of therapy with retinoidcompounds, there occurs occasionally a serious medical condition causedby vitamin A or vitamin A precursor overdose, resulting either from theexcessive intake of vitamin supplements or the ingestion of liver ofcertain fish and animals that contain high levels of the vitamin. Thechronic or acute toxicities observed with hypervitaminosis A syndromeinclude headache, skin peeling, bone toxicity, dyslipidemias, etc. Inrecent years, it has become apparent that the toxicities observed withvitamin A analogs, i.e., retinoids, essentially recapitulate those ofhypervitaminosis A syndrome, suggesting a common biological cause, i.e.,RAR activation. These toxicities are presently treated mainly bysupportive measures and by abstaining from further exposure to thecausative agent, whether it be liver, vitamin supplements, or retinoids.While some of the toxicities resolve with time, others (e.g., prematureepiphyseal plate closure) are permanent.

Generally speaking, specific antidotes are the best treatment forpoisoning by pharmacological agents, but only about two dozen chemicalsor classes of chemicals out of thousands in existence have specificknown antidotes. A specific antidote would clearly be of value in thetreatment of hypervitaminosis A and retinoid toxicity. Indeed, asincreasingly potent retinoids are used clinically, a specific antidotefor retinoid poisoning could be life saving.

SUMMARY OF THE INVENTION

The present invention covers compounds of Formula 1 ##STR1##

wherein X is S, O, NR' where R' is H or alkyl of 1 to 6 carbons, or

X is C(R₁)₂ !_(n) where R₁ is independently H or alkyl of 1 to 6carbons, and n is an integer between 0 and 2;

R₂ is hydrogen, lower alkyl of 1 to 6 carbons, F, Cl, Br, I, CF₃, fluorosubstituted alkyl of 1 to 6 carbons, OH, SH, alkoxy of 1 to 6 carbons,or alkylthio of 1 to 6 carbons;

R₃ is hydrogen, lower alkyl of 1 to 6 carbons or F;

m is an integer having the value of 0-3;

o is an integer having the value of 0-3;

Z is --C═C--,

--N═N--,

--N═CR₁ --,

--CR₁ ═N,

--(CR₁ ═CR₁)_(n) --where n' is an integer having the value 0-5,

--CO--NR₁ --,

--CS--NR₁ --,

--NR₁ --CO,

--NR₁ --CS,

--COO--,

--OCO--;

--CSO--;

--OCS--;

Y is a phenyl or naphthyl group, or heteroaryl selected from a groupconsisting of pyridyl, thienyl, furyl, pyridazinyl, pyrimidinyl,pyrazinyl, thiazolyl, oxazolyl, imidazolyl and pyrrazolyl, said phenyland heteroaryl groups being optionally substituted with one or two R₂groups, or

when Z is --(CR₁ ═CR₁)_(n) -- and n' is 3, 4 or 5 then Y represents adirect valence bond between said (CR₂ ═CR₂)_(n), group and B;

A is (CH₂)_(q) where q is 0-5, lower branched chain alkyl having 3-6carbons, cycloalkyl having 3-6 carbons, alkenyl having 2-6 carbons and 1or 2 double bonds, alkynyl having 2-6 carbons and 1 or 2 triple bonds;

B is hydrogen, COOH or a pharmaceutically acceptable salt thereof,COOR₈, CONR₉ R₁₀, --CH₂ OH, CH₂ OR₁₁, CH₂ OCOR₁₁, CHO, CH(OR₁₂)₂, CHOR₁₃O, --COR₇, CR₇ (OR₁₂)₂, CR₇ OR₁₃ O, or tri-lower alkylsilyl, where R₇ isan alkyl, cycloalkyl or alkenyl group containing 1 to 5 carbons, R₈ isan alkyl group of 1 to 10 carbons or trimethylsilylalkyl where the alkylgroup has 1 to 10 carbons, or a cycloalkyl group of 5 to 10 carbons, orR₈ is phenyl or lower alkylphenyl, R₉ and R₁₀ independently arehydrogen, an alkyl group of 1 to 10 carbons, or a cycloalkyl group of5-10 carbons, or phenyl or lower alkylphenyl, R₁₁ is lower alkyl, phenylor lower alkylphenyl, R₁₂ is lower alkyl, and R₁₃ is divalent alkylradical of 2-5 carbons, and

R₁₄ is (R₁₅)_(r) -phenyl, (R₁₅)_(r) -naphthyl, or (R₁₅)_(r) - heteroarylwhere the heteroaryl group has 1 to 3 heteroatoms selected from thegroup consisting of O, S and N, r is an integer having the values of0-5, and

R₁₅ is independently H, F, Cl, Br, I, NO₂, N(R₈)₂, N(R₈)COR₈, NR₈CON(R₈)₂, OH, OCOR₈, OR₈, CN, an alkyl group having 1 to 10 carbons,fluoro substituted alkyl group having 1 to 10 carbons, an alkenyl grouphaving 1 to 10 carbons and 1 to 3 double bonds, alkynyl group having 1to 10 carbons and 1 to 3 triple bonds, or a trialkylsilyl ortrialkylsilyloxy group where the alkyl groups independently have 1 to 6carbons.

The present invention further covers compounds of Formula 101 ##STR2##

wherein X is S, O, NR' where R' is H or alkyl of 1 to 6 carbons, or

X is C(R₁)₂ !_(n) where R₁ is independently H or alkyl of 1 to 6carbons, and n is an integer between 0 and 2;

R₂ is hydrogen, lower alkyl of 1 to 6 carbons, F, Cl, Br, I, CF₃, fluorosubstituted alkyl of 1 to 6 carbons, OH, SH, alkoxy of 1 to 6 carbons,or alkylthio of 1 to 6 carbons;

R₃ is hydrogen, lower alkyl of 1 to 6 carbons or F;

m is an integer having the value of 0-3;

o is an integer having the value of 0-3;

Y is a phenyl or naphthyl group, or heteroaryl selected from a groupconsisting of pyridyl, thienyl, furyl, pyridazinyl, pyrimidinyl,pyrazinyl, thiazolyl, oxazolyl, imidazolyl and pyrrazolyl, said phenyland heteroaryl groups being optionally substituted with one or two R₂groups;

A is (CH₂)_(q) where q is 0-5, lower branched chain alkyl having 3-6carbons, cycloalkyl having 3-6 carbons, alkenyl having 2-6 carbons and 1or 2 double bonds, alkynyl having 2-6 carbons and 1 or 2 triple bonds;

B is hydrogen, COOH or a pharmaceutically acceptable salt thereof,COOR₈, CONR₉ R₁₀, --CH₂ OH, CH₂ OR₁₁, CH₂ OCOR₁₁, CHO, CH(OR₁₂)₂, CHOR₁₃O, --COR₇, CR₇ (OR₁₂)₂, CR₇ OR₁₃ O, or tri-lower alkylsilyl, where R₇ isan alkyl, cycloalkyl or alkenyl group containing 1 to 5 carbons, R₈ isan alkyl group of 1 to 10 carbons or trimethylsilylalkyl where the alkylgroup has 1 to 10 carbons, or a cycloalkyl group of 5 to 10 carbons, orR₈ is phenyl or lower alkylphenyl, R₉ and R₁₀ independently arehydrogen, an alkyl group of 1 to 10 carbons, or a cycloalkyl group of5-10 carbons, or phenyl or lower alkylphenyl, R₁₁ is lower alkyl, phenylor lower alkylphenyl, R₁₂ is lower alkyl, and R₁₃ is divalent alkylradical of 2-5 carbons, and

R₁₄ is (R₁₅)_(r) -phenyl, (R₁₅)_(r) -naphthyl, or (R₁₅)_(r) - heteroarylwhere the heteroaryl group has 1 to 3 heteroatoms selected from thegroup consisting of O, S and N, r is an integer having the values of0-5, and

R₁₅ is independently H, F, Cl, Br, I, NO₂, N(R₈)₂, N(R₈)COR₈, NR₈CON(R₈)₂, OH, OCOR₈, OR₈, CN, an alkyl group having 1 to 10 carbons,fluoro substituted alkyl group having 1 to 10 carbons, an alkenyl grouphaving 1 to 10 carbons and 1 to 3 double bonds, alkynyl group having 1to 10 carbons and 1 to 3 triple bonds, or a trialkylsilyl ortrialkylsilyloxy group where the alkyl groups independently have 1 to 6carbons;

R₁₆ is H, lower alkyl of 1 to 6 carbons;

R₁₇ is H, lower alkyl of 1 to 6 carbons, OH or OCOR₁₁, and

p is zero or 1, with the proviso that when p is 1 then there is no R₁₇substituent group, and m is an integer between 0 and 2.

The compounds of the present invention are useful for preventing certainundesired side effects of retinoids which are administered for thetreatment or prevention of certain diseases or conditions. For thispurpose the compounds of the invention may be coadministered withretinoids. The compounds of the present invention are also useful in thetreatment of acute or chronic toxicity resulting from overdose orpoisoning by retinoid drugs or Vitamin A.

The present invention additionally relates to the use of RAR antagonistsfor blocking all or some RAR receptor sites in biological systems,including mammals, to prevent or diminish action of RAR agonists on saidreceptor sites. More particularly, the present invention relates to theuse of RAR antagonists for (a) the prevention and (b) the treatment ofretinoid (including vitamin A or vitamin A precursor) chronic or acutetoxicity and side effects of retinoid therapy.

In one particular aspect of the present invention, there is provided amethod of treating a pathological condition in a mammal. The conditionstreated are associated with a retinoic acid receptor activity. Thismethod involves administering to the mammal a retinoid antagonist ornegative hormone capable of binding to one of the following retinoicacid receptor subtypes: RAR.sub.α, RAR.sub.β and RAR.sub.γ. Theantagonist or negative hormone is administered in an amountpharmaceutically effective to provide a therapeutic benefit against thepathological condition in the mammal.

As an antidote to acute or chronic retinoid or vitamin A poisoning theRAR antagonist can be administered to a mammal enterally, i.e.,intragastric intubation or food/water admixture, or parenterally, e.g.,intraperitoneally, intramuscularly, subcutaneously, topically, etc. Theonly requirement for the route of administration is that it must allowdelivery of the antagonist to the target tissue. The RAR antagonist canbe formulated by itself or in combination with excipients. The RARantagonist need not be in solution in the formulation, e.g., in the caseof enteral use.

As an adjunct to therapy with retinoids and in order to prevent one ormore side effects of the retinoid drug which is administered, the RARantagonist can similarly be administered enterally or parenterally. TheRAR antagonist and RAR agonist need not be administered by the sameroute of administration. The key is that sufficient quantities of theRAR antagonist be present continuously in the tissue of interest duringexposure to the RAR agonist. For the prevention of retinoid toxicity, itis best that the RAR antagonist be administered concurrently or prior totreatment with the RAR agonist. In many situations the RAR antagonistwill be administered by a different route than the agonist. For exampleundesirable skin effects of an enterally administered retinoid may beprevented or ameliorated by an RAR antagonist which is administeredtopically.

Another aspect of the present invention is a method of identifyingretinoid negative hormones. The method includes the following steps:obtaining transfected cells containing a reporter gene transcriptionallyresponsive to binding of a recombinant retinoid receptor, therecombinant retinoid receptor having at least protein domains locatedC-terminal to a DNA binding domain of an intact retinoid receptor,measuring a basal level of reporter gene expression in untreatedtransfected cells, the untreated transfected cells being propagated inthe absence of an added retinoid, treating the transfected cells with aretinoid compound to be tested for negative hormone activity, measuringa level of reporter gene expression in treated cells, comparing thelevels of reporter gene expression measured in treated cells anduntreated cells, and identifying as retinoid negative hormones thoseretinoid compounds producing a lower level of reporter gene expressionin treated cells compared with the basal level of reporter geneexpression measured in untreated cells. In certain preferred embodimentsof this method the intact receptor is an RAR-α, RAR-β or RAR-γ subtype.In other embodiments, the intact receptor is an RXR-α, RXR-β or RXR-γsubtype. The recombinant receptor can also be either a recombinant RARor RXR receptor. In some embodiments, the recombinant receptor is achimeric retinoid receptor having a constitutive transcription activatordomain. Such a constitutive transcription activator domain can comprisea plurality of amino acids having a net negative charge or have an aminoacid sequence of a viral transcription activator domain, such as theherpes simplex virus VP-16 transcription activator domain. Inembodiments in which the constitutive transcription activator domain hasa net negative charge, the retinoid receptor can be recombinant and havedeleted therefrom a DNA binding domain, such as a DNA binding domainspecific for a cis-regulatory element other than a retinoic acidresponsive element. These elements include an estrogen responsiveelement. The transfected cell is preferably propagated in a growthmedium substantially depleted of endogenous retinoids, such as one thatincludes activated charcoal-extracted serum. In this method, thereporter gene can be the luciferase gene, in which case, the measuringsteps can involve luminometry. The reporter gene can also be theβ-galactosidase gene, in which case the measuring steps would involve aβ-galactosidase assay. The transfected cell can be a transfectedmammalian cell, such as a Green monkey cell or a human cell.

Another aspect of the present invention is a method of potentiating apharmacologic activity of a steroid superfamily receptor agonistadministered to a mammal. This method involves coadministering to themammal with the steroid superfamily receptor agonist a compositioncomprising a pharmaceutically effective dose of a retinoid negativehormone to potentiate the pharmacologic activity of the steroidsuperfamily receptor agonist. The pharmacologic activity is measurablein a reporter gene trans-activation assay in vitro, such as by measuringanti-AP-1 activity. The pharmacologic activity to be potentiated can bean antiproliferative activity, such as activity of the type measurablein retinal pigment epithelium. The steroid superfamily receptor agonistcan be any of the following: a retinoid receptor agonist, a vitamin Dreceptor agonist, a glucocorticoid receptor agonist, a thyroid hormonereceptor agonist, a peroxisome proliferator-activated receptor agonistor an estrogen receptor agonist. The retinoid receptor agonist can be anRAR agonist, such as all-trans-retinoic acid or 13-cis retinoic acid.The retinoid receptor agonist can also be an RXR agonist. A preferredvitamin D receptor agonist is 1,25-dihydroxyvitamin D₃. A preferredglucocorticoid receptor agonist is dexamethasone. A preferred thyroidhormone receptor agonist is 3,3',5-triiodothyronine. The retinoidnegative hormone is an RAR-specific retinoid negative hormone, whichpreferably has a dissociation constant less than or approximately equalto 30 nM. Example of the RAR-specific retinoid negative hormone includeAGN 193109, AGN 193385, AGN 193389 and AGN 193871. The compositioncomprising a pharmaceutically effective dose of a retinoid negativehormone can be coadministered at the same time as the steroidsuperfamily agonist and be combined prior to coadministration. These canalso be coadministered as separate compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of AGN 193109.

FIGS. 2A-2F are a series of line graphs showing that AGN 193109inhibited ATRA-dependent transactivation at the RARs. FIGS. 2A and 2Brepresent activity at the RAR-α receptor; FIGS. 2C and 2D representactivity at the RAR-β receptor; FIGS. 2E and 2F represent activity atthe RAR-γ receptor. In FIGS. 2A, 2C and 2E, open squares representretinoic acid treatment and filled circles represent AGN 193109treatment. In FIGS. 2B, 2D and 2F the single lines represent luciferaseactivity measured after treatment with 10⁻⁸ M ATRA and variableconcentrations of AGN 193109.

FIGS. 3A and 3B are line graphs representing luciferase activitydetected in CV-1 cells transfected with reporter plasmid ERE-tk-Luc andexpression plasmid ER-RAR-α and stimulated with ATRA (FIG. 3A) or AGN193109 (FIG. 3B) at various concentrations. Data points represent themean ±SEM of three independent luciferase determinations. The results oftransfections carried out using different amounts of co-transfectedER-RAR-α (0.05, 0.1 and 0.2 μg/well) are indicated in each figure.

FIGS. 4A and 4B are line graphs representing luciferase activity in CV-1cells transfected with reporter plasmid ERE-tk-Luc and expressionplasmid ER-RAR-β and stimulated with ATRA (FIG. 4A) or AGN 193109 (FIG.4B) at various concentrations. Data points represent the mean ±SEM ofthree independent luciferase determinations. The results oftransfections carried out using different amounts of co-transfectedER-RAR-β (0.05, 0.1 and 0.2 μg/well) are indicated in each figure.

FIGS. 5A and SB are line graphs representing luciferase activitydetected in CV-1 cells transfected with reporter plasmid ERE-tk-Luc andexpression plasmid ER-RAR-γ and stimulated with ATRA (FIG. 5A) or AGN193109 (FIG. 5B) at various concentrations. Data points represent themean ±SEM of three independent luciferase determinations. The results oftransfections carried out using different amounts of co-transfectedER-RAR-γ (0.05, 0.1 and 0.2 μg/well) are indicated in each figure.

FIG. 6 shows ATRA and AGN 193109 dose responses of CV-1 cellscotransfected with the ERE-tk-Luc reporter plasmid and either theER-RXR-α chimeric receptor expression plasmid alone, or in combinationwith the RAR-γ-VP-16 expression plasmid. ER-RXR-α cotransfected cellswere treated with ATRA (square) and AGN 193109 (diamond). Cellscotransfected with the combination of ER-RXR-α and RAR-γ-VP-16 weretreated with ATRA (circle) or AGN 193109 (triangle).

FIG. 7 shows a line graph representing luciferase activity measurementsrecorded in lysates of CV-1 cells transfected with the ERE-tk-Lucreporter and ER-RAR-γ expression construct and then treated with ATRA at10⁻⁸ M and the test compounds at the concentrations indicated on thehorizontal axis. The test compounds were AGN 193109 (square), AGN 193357(open diamond), AGN 193385 (circle), AGN 193389 (triangle), AGN 193840(hatched square) and AGN 192870 (filled diamond).

FIG. 8 shows a line graph representing luciferase activity measurementsrecorded in lysates of CV-1 cells transfected with the ERE-tk-Lucreporter and RAR-γ-VP-16 and ER-RXR-α expression constructs and thentreated with the test compounds at the concentrations indicated on thehorizontal axis. The test compounds were ATRA (open square), AGN 193109(open circle), AGN 193174 (open triangle), AGN 193199 (hatched square),AGN 193385 (hatched circle), AGN 193389 (inverted triangle), AGN 193840(diagonally filled square) and AGN 193871 (half-filled diamond).

FIGS. 9A, 9B and 9C schematically diagram a mechanism whereby AGN 193109can modulate the interaction between the RAR (shaded box) and negativecoactivator proteins (-) illustrated in the context of a transactivationassay. FIG. 9A shows that negative coactivator proteins and positivecoactivator proteins (+) are in a binding equilibrium with the RAR. Inthe absence of a ligand, basal level transcription of the reporter generesults. As illustrated in FIG. 9B, addition of an RAR agonist promotesthe association of positive coactivator proteins with the RAR andresults in upregulated reporter gene transcription. As illustrated inFIG. 9C, addition of AGN 193109 promotes the association of negativecoactivator proteins with the RAR and prevents reporter genetranscription.

FIG. 10 is a bar graph showing the inhibition of TPA-induced Str-AP1-CATexpression as a function of AGN 191183 concentration (10⁻¹⁰ to 10⁻¹² M)with the AGN 193109 concentration held constant at 10⁻⁸ M. Results fromtrials conducted with AGN 191183 alone are shown as hatched bars whilestripped bars represent the results from treatment with the combinationof AGN 193109 and AGN 191183.

FIG. 11 schematically diagrams a mechanism whereby AGN 193109 canpotentiate the activities of RARs and other nuclear receptor familymembers. As illustrated in the diagram, introduced RARs (open rectangleshaving AB-C-DEF domains) have increased sensitivity to RAR ligands inthe anti-AP1 assay because the negative coactivator protein (ncp),present in limiting supply, is sequestered onto RARs thereby leading totwo populations: RAR+ncp and RAR-ncp. RAR-ncp has increased sensitivityto ligands. Non-RAR nuclear factors (shaded rectangles having AB-C-DEFdomains) have increased sensitivity to cognate ligands because ncp hasbeen sequestered to the RAR by the activity of AGN 193109. The modulardomains of the nuclear receptors are designated using standardnomenclature as "AB" (ligand independent transactivation domain), "C"(DNA binding domain), and "DEF" (ligand regulated transactivation domainand dimerization domain.

FIG. 12 is a line graph showing the effect of AGN 193109 on the1,25-dihydroxyvitamin D₃ dose response in CV-1 cells transfected withthe MTV-DR3-Luc reporter plasmid. Transfectants were treated with1,25-dihydroxyvitamin D₃ (filled square), 1,25-dihydroxyvitamin D₃ and10⁻⁸ M AGN 193109 (filled triangle), and 1,25-dihydroxyvitamin D₃ and10⁻⁷ M AGN 193109 (filled circle).

FIG. 13 is a bar graph showing the effect of AGN 193109 (10 nM)coadministration on 1,25-dihydroxyvitamin D₃ -mediated inhibition of TPAinduced Str-AP1-CAT activity. Filled bars represent inhibition of CATactivity in transfected cells treated with 1,25-dihydroxyvitamin D₃alone. Open bars represent inhibition of CAT activity in transfectedcells treated with the combination of 1,25-dihydroxyvitamin D₃ and AGN193109.

FIG. 14 is a line graph showing the effect of AGN 193109 alone and incombination with AGN 191183 on HeLa cells cotransfected with RAR-γ andthe RAR responsive MTV-TREp-Luc reporter construct. Drug treatmentsillustrated in the graph are: AGN 193109 alone (square), AGN 193109 incombination with AGN 191183 at 10⁻¹⁰ M (diamond) and AGN 193109 incombination with AGN 191183 at 10⁻⁹ M.

FIG. 15 is a line graph showing that ECE16-1 cells proliferated inresponse to EGF (filled square) but not in response to defined mediumalone (open circle). Cells treated with AGN 193109 alone are representedby the filled triangle. The filled circles represent results obtainedfor cells treated with 10 nM AGN 191183 and 0-1000 nM AGN 193109.

FIG. 16 is a bar graph showing the effect of AGN 193109 on theproliferation of CaSki cells in the presence or absence of the AGN191183 retinoid agonist. All sample groups received 20 ng/ml ofepidermal growth factor (EGF) with the exception of the samplepropagated in defined medium (DM) alone (open bar). Stripped barsrepresent samples propagated in the absence of AGN 193109. Filled barsrepresent samples propagated in the presence of 1000 nM AGN 193109. Theconcentrations of AGN 191183 used in the procedure are shown on thehorizontal axis.

FIG. 17 is a dose response curve showing that AGN 193109 potentiated theantiproliferative activity of ATRA on retinal pigment epithelium (RPE)cells. Samples treated with ATRA alone are represented by filledsquares. Samples treated with the combination of ATRA and AGN 193109(10⁻⁷ M) are represented by filled circles. The ATRA concentration usedfor treating the various samples is given on the horizontal axis.

FIG. 18 is a dose response curve showing that both 13-cis-RA and ATRAinhibited RPE cell growth, and that AGN 193109 potentiated theantiproliferative activity of 13-cis-RA. The various sample treatmentsshown in the dose response included 13-cis-RA alone (filled square),13-cis-RA in combination with AGN 193109 (10⁻⁶ M) (filled circle),13-cis-RA in combination with AGN 193109 (10⁻⁸ M) (filled triangle), andATRA (filled diamond). The concentrations of 13-cis-RA and ATRA used inthe sample treatments are shown on the horizontal axis.

FIG. 19 is a dose response curve showing that AGN 193109 potentiated theantiproliferative activity of dexamethasone in primary RPE cellcultures. The various sample treatments shown in the dose responseincluded ATRA (filled square), dexamethasone alone (filled circle),dexamethasone in combination with AGN 193109 (10⁻⁸ M) (filled triangle),and dexamethasone in combination with AGN 193109 (10⁻⁶ M) (filleddiamond). The concentrations of dexamethasone and ATRA used in thesample treatments are shown on the horizontal axis.

FIG. 20 is a dose response curve showing that AGN 193109 potentiated theantiproliferative activity of thyroid hormone (T3) in primary RPE cellcultures. The various sample treatments shown in the dose responseincluded ATRA (filled square), T3 alone (filled circle), T3 incombination with AGN 193109 (10⁻⁸ M) (filled triangle), T3 incombination with AGN 193109 (10⁻⁶ M) (filled diamond). Theconcentrations of T3 and ATRA used in the sample treatments are shown onthe horizontal axis.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

For the purposes of the present invention, an RAR antagonist is definedas a chemical that binds to one or more of the RAR subtypes with a K_(d)of less than 1 micromolar (K_(d) <1 μM) but which does not causesignificant transcriptional activation of that RAR subtypes-regulatedgenes in a receptor co-transfection assay. Conventionally, antagonistsare chemical agents that inhibit the activities of agonists. Thus, theactivity of a receptor antagonist is conventionally measured by virtueof its ability to inhibit the activity of an agonist.

An RAR agonist is defined as a chemical that binds to one or more RARreceptor subtype with K_(d) of less than 1 micromol (K_(d) <1 μM) andcauses transcriptional activation of that RAR-subtype-regulated genes ina receptor co-transfection assay. The term "RAR agonist" includeschemicals that may bind and/or activate other receptors in addition toRARs, e.g., RXR receptors.

As used herein, a negative hormone or inverse agonist is a ligand for areceptor which causes the receptor to adopt an inactive state relativeto a basal state occurring in the absence of any ligand. Thus, while anantagonist can inhibit the activity of an agonist, a negative hormone isa ligand that can alter the conformation of the receptor in the absenceof an agonist. The concept of a negative hormone or inverse agonist hasbeen explored by Bond et al. in Nature 374: 272 (1995). Morespecifically, Bond et al. have proposed that unliganded β₂ -adrenoceptorexists in an equilibrium between an inactive conformation and aspontaneously active conformation. Agonists are proposed to stabilizethe receptor in an active conformation. Conversely, inverse agonists arebelieved to stabilize an inactive receptor conformation. Thus, while anantagonist manifests its activity by virtue of inhibiting an agonist, anegative hormone can additionally manifest its activity in the absenceof an agonist by inhibiting the spontaneous conversion of an unligandedreceptor to an active conformation. Only a subset of antagonists willact as negative hormones. As disclosed herein, AGN 193109 is both anantagonist and a negative hormone. To date, no other retinoids have beenshown to have negative hormone activity.

As used herein, coadministration of two pharmacologically activecompounds refers to the delivery of two separate chemical entities,whether in vitro or in vivo. Coadministration refers to the simultaneousdelivery of separate agents; to the simultaneous delivery of a mixtureof agents; as well as to the delivery of one agent followed by deliveryof the second agent. In all cases, agents that are coadministered areintended to work in conjunction with each other.

The term alkyl refers to and covers any and all groups which are knownas normal alkyl, branched-chain alkyl and cycloalkyl. The term alkenylrefers to and covers normal alkenyl, branch chain alkenyl andcycloalkenyl groups having one or more sites of unsaturation. Similarly,the term alkynyl refers to and covers normal alkynyl, and branch chainalkynyl groups having one or more triple bonds.

Lower alkyl means the above-defined broad definition of alkyl groupshaving 1 to 6 carbons in case of normal lower alkyl, and as applicable 3to 6 carbons for lower branch chained and cycloalkyl groups. Loweralkenyl is defined similarly having 2 to 6 carbons for normal loweralkenyl groups, and 3 to 6 carbons for branch chained and cyclo- loweralkenyl groups. Lower alkynyl is also defined similarly, having 2 to 6carbons for normal lower alkynyl groups, and 4 to 6 carbons for branchchained lower alkynyl groups.

The term "ester" as used here refers to and covers any compound fallingwithin the definition of that term as classically used in organicchemistry. It includes organic and inorganic esters. Where B (of Formula1 or Formula 101) is --COOH, this term covers the products derived fromtreatment of this function with alcohols or thiols preferably withaliphatic alcohols having 1-6 carbons. Where the ester is derived fromcompounds where B is --CH₂ OH, this term covers compounds derived fromorganic acids capable of forming esters including phosphorous based andsulfur based acids, or compounds of the formula --CH₂ OCORH₁₁ where R₁₁is any substituted or unsubstituted aliphatic, aromatic, heteroaromaticor aliphatic aromatic group, preferably with 1-6 carbons in thealiphatic portions.

Unless stated otherwise in this application, preferred esters arederived from the saturated aliphatic alcohols or acids of ten or fewercarbon atoms or the cyclic or saturated aliphatic cyclic alcohols andacids of 5 to 10 carbon atoms. Particularly preferred aliphatic estersare those derived from lower alkyl acids and alcohols. Also preferredare the phenyl or lower alkyl phenyl esters.

Amides has the meaning classically accorded that term in organicchemistry. In this instance it includes the unsubstituted amides and allaliphatic and aromatic mono- and di- substituted amides. Unless statedotherwise in this application, preferred amides are the mono- anddi-substituted amides derived from the saturated aliphatic radicals often or fewer carbon atoms or the cyclic or saturated aliphatic-cyclicradicals of 5 to 10 carbon atoms. Particularly preferred amides arethose derived from substituted and unsubstituted lower alkyl amines.Also preferred are mono- and disubstituted amides derived from thesubstituted and unsubstituted phenyl or lower alkylphenyl amines.Unsubstituted amides are also preferred.

Acetals and ketals include the radicals of the formula-CK where K is(--OR)₂. Here, R is lower alkyl. Also, K may be --OR₇ O-- where R₇ islower alkyl of 2-5 carbon atoms, straight chain or branched.

A pharmaceutically acceptable salt may be prepared for any compounds inthis invention having a functionality capable of forming a salt, forexample an acid functionality. A pharmaceutically acceptable salt is anysalt which retains the activity of the parent compound and does notimpart any deleterious or untoward effect on the subject to which it isadministered and in the context in which it is administered.

Pharmaceutically acceptable salts may be derived from organic orinorganic bases. The salt may be a mono or polyvalent ion. Of particularinterest are the inorganic ions, sodium, potassium, calcium, andmagnesium. Organic salts may be made with amines, particularly ammoniumsalts such as mono-, di- and trialkyl amines or ethanol amines. Saltsmay also be formed with caffeine, tromethamine and similar molecules.Where there is a nitrogen sufficiently basic as to be capable of formingacid addition salts, such may be formed with any inorganic or organicacids or alkylating agent such as methyl iodide. Preferred salts arethose formed with inorganic acids such as hydrochloric acid, sulfuricacid or phosphoric acid. Any of a number of simple organic acids such asmono-, di- or tri- acid may also be used.

Some of the compounds of the present invention may have trans and cis (Eand Z) isomers. In addition, the compounds of the present invention maycontain one or more chiral centers and therefore may exist inenantiomeric and diastereomeric forms. The scope of the presentinvention is intended to cover all such isomers per se, as well asmixtures of cis and trans isomers, mixtures of diastereomers and racemicmixtures of enantiomers (optical isomers) as well. In the presentapplication when no specific mention is made of the configuration (cis,trans or R or S) of a compound (or of an asymmetric carbon) then amixture of such isomers, or either one of the isomers is intended.

Aryl Substituted Benzopyran, Benzothiopyran, 1,2-Dihydroquinoline and5,6-Dihydronaphthalene Derivatives Having Retinoid Antagonist LikeBiological Activity

With reference to the symbol Y in Formula 1, the preferred compounds ofthe invention are those where Y is phenyl, pyridyl, thienyl or furyl.Even more preferred are compounds where Y is phenyl or pyridyl, andstill more preferred where Y is phenyl. As far as substitutions on the Y(phenyl) and Y (pyridyl) groups are concerned, compounds are preferredwhere the phenyl group is 1,4 (para) substituted by the Z and A-Bgroups, and where the pyridine ring is 2,5 substituted by the Z and A-Bgroups. (Substitution in the 2,5 positions in the "pyridine"nomenclature corresponds to substitution in the 6-position in the"nicotinic acid" nomenclature.) In the preferred compounds of theinvention either there is no optional R₂ substituent on the Y group, orthe optional R₂ substituent is fluoro (F).

The A-B group of the preferred compounds is (CH₂)_(n) --COOH or(CH₂)_(n) --COOR₈, where n and R₈ are defined as above. Even morepreferably n is zero and R₈ is lower alkyl, or n is zero and B is COOHor a pharmaceutically acceptable salt thereof.

In the majority of the presently preferred examples of compounds of theinvention X is C(R₁)₂ !_(n) where n is 1. Nevertheless, compounds wheren is zero (indene derivatives) and where X is S or O (benzothiopyran andbenzopyran derivatives) are also preferred. When X is C(R₁)₂ !_(n) and nis 1, then R₁ preferably is alkyl of 1 to 6 carbons, even morepreferably methyl.

The R₂ group attached to the aromatic portion of thetetrahydronaphthalene, benzopyran, benzothiopyran or dihydroquinolinemoiety of the compounds of Formula 1 is preferably H, F or CF₃. R₃ ispreferably hydrogen or methyl, even more preferably hydrogen.

Referring now to the group Z in the compounds of the invention and shownin Formula 1, in a plurality of preferred examples Z represents anacetylenic linkage (Z=--C.tbd.C--). However, the "linker group" Z isalso preferred as a diazo group (Z=-N═N--), as an olefinic orpolyolefinic group (Z----(CR₁ ═CR₁)_(n) --), as an ester (Z═--COO--),amide (Z=--CO--NR₂ --) or thioamide (Z═--CS--NR₂ --) linkage.

Referring now to the R₁₄ group, compounds are preferred where R₁₄ isphenyl, 2-pyridyl, 3-pyridyl, 2- thienyl, and 2-thiazolyl. The R₁₅ group(substituent of the R₁₄ group) is preferably H, lower alkyl,trifluoromethyl, chlorine, lower alkoxy or hydroxy.

The presently most preferred compounds of the invention are shown inTable 1 with reference to Formula 2, Formula 3, Formula 4, Formula 5,and Formula 5a. ##STR3##

                                      TABLE 1                                     __________________________________________________________________________    Compound                                                                      #     Formula                                                                           R.sub.14 * Z      R.sub.2 *                                                                        R.sub.8 *                                      __________________________________________________________________________    1     2   4-methylphenyl                                                                           --C.tbd.C--                                                                          H  Et                                             1a    2   phenyl     --C.tbd.C--                                                                          H  Et                                             2     2   3-methylphenyl                                                                           --C.tbd.C--                                                                          H  Et                                             3     2   2-methylphenyl                                                                           --C.tbd.C--                                                                          H  Et                                             4     2   3,5-dimethylphenyl                                                                       --C.tbd.C--                                                                          H  Et                                             5     2   4-ethylphenyl                                                                            --C.tbd.C--                                                                          H  Et                                             6     2   4-t-butylphenyl                                                                          --C.tbd.C--                                                                          H  Et                                             7     2   4-chlorophenyl                                                                           --C.tbd.C--                                                                          H  Et                                             8     2   4-methoxyphenyl                                                                          --C.tbd.C--                                                                          H  Et                                             9     2   4-trifluoromethylphenyl                                                                  --C.tbd.C--                                                                          H  Et                                             10    2   2-pyridyl  --C.tbd.C--                                                                          H  Et                                             11    2   3-pyridyl  --C.tbd.C--                                                                          H  Et                                             12    2   2-methyl-5-pyridyl                                                                       --C.tbd.C--                                                                          H  Et                                             13    2   3-hydroxyphenyl                                                                          --C.tbd.C--                                                                          H  Et                                             14    2   4-hydroxy phenyl                                                                         --C.tbd.C--                                                                          H  Et                                             15    2   5-methyl-2-thiazolyl                                                                     --C.tbd.C--                                                                          H  Et                                             15a   2   2-thiazolyl                                                                              --C.tbd.C--                                                                          H  Et                                             16    2   4-methyl-2-thiazolyl                                                                     --C.tbd.C--                                                                          H  Et                                             17    2   4,5-dimethyl-2-thiazolyl                                                                 --C.tbd.C--                                                                          H  Et                                             18    2   2-methyl-5-pyridyl                                                                       --C.tbd.C--                                                                          H  H                                              19    2   2-pyridyl  --C.tbd.C--                                                                          H  H                                              20    2   3-methylphenyl                                                                           --C.tbd.C--                                                                          H  H                                              21    2   4-ethylphenyl                                                                            --C.tbd.C--                                                                          H  H                                              22    2   4-methoxyphenyl                                                                          --C.tbd.C--                                                                          H  H                                              23    2   4-trifluoromethylphenyl                                                                  --C.tbd.C--                                                                          H  H                                              24    2   3,5-dimethylphenyl                                                                       --C.tbd.C--                                                                          H  H                                              25    2   4-chlorophenyl                                                                           --C.tbd.C--                                                                          H  H                                              26    2   3-pyridyl  --C.tbd.C--                                                                          H  H                                              27    2   2-methylphenyl                                                                           --C.tbd.C--                                                                          H  H                                              28    2   3-hydroxyphenyl                                                                          --C.tbd.C--                                                                          H  H                                              29    2   4-hydroxyphenyl                                                                          --C.tbd.C--                                                                          H  H                                              30    2   5-methyl-2-thiazolyl                                                                     --C.tbd.C--                                                                          H  H                                              30a   2   2-thiazolyl                                                                              --C.tbd.C--                                                                          H  H                                              31    2   4-methyl-2-thiazolyl                                                                     --C.tbd.C--                                                                          H  H                                              32    2   4,5-dimethyl-2-thiazolyl                                                                 --C.tbd.C--                                                                          H  H                                              33    2   5-methyl-2-thienyl                                                                       --C.tbd.C--                                                                          H  Et                                             33a   2   2-thienyl  --C.tbd.C--                                                                          H  Et                                             34    2   5-methyl-2-thienyl                                                                       --C.tbd.C--                                                                          H  H                                              34a   2   2-thienyl  --C.tbd.C--                                                                          H  H                                              35    2   4-methylphenyl                                                                           --CONH--                                                                             H  Et                                             36    2   4-methylphenyl                                                                           --CONH--                                                                             H  H                                              37    2   4-methylphenyl                                                                           --COO--                                                                              H  Et                                             38    2   4-methylphenyl                                                                           --COO--                                                                              H  (CH.sub.2).sub.2 Si(CH.sub.3)                  39    2   4-methylphenyl                                                                           --COO--                                                                              H  H                                              40    2   4-methylphenyl                                                                           --CONH--                                                                             F  Et                                             41    2   4-methylphenyl                                                                           --CONH--                                                                             F  H                                              42    2   4-methylphenyl                                                                           --CSNH--                                                                             H  Et                                             43    2   4-methylphenyl                                                                           --CSNH--                                                                             H  H                                              44    2   4-methylphenyl                                                                           --CH═CH--                                                                        H  Et                                             45    2   4-methylphenyl                                                                           --CH═CH--                                                                        H  H                                              46a   2   4-methylphenyl                                                                           --N═N--                                                                          H  Et                                             46b   2   4-methylphenyl                                                                           --N═N--                                                                          H  H                                              47    3   4-methylphenyl                                                                           --C.tbd.C--                                                                          H  Et                                             48    3   4-methylphenyl                                                                           --C.tbd.C--                                                                          H  H                                              49    4   4-methylphenyl                                                                           --C.tbd.C--                                                                          H  Et                                             50    4   4-methylphenyl                                                                           --C.tbd.C--                                                                          H  H                                              51    5   4-methylphenyl                                                                           --     -- Et                                             52    5   4-methylphenyl                                                                           --     -- H                                              60    2   4-methylphenyl                                                                           --C.tbd.C--                                                                          H  H                                              60a   2   phenyl     --C.tbd.C--                                                                          H  H                                              61    2   4-t-butylphenyl                                                                          --C.tbd.C--                                                                          H  H                                              62    2   4-methylphenyl                                                                           --CSNH--                                                                             F  Et                                             63    2   4-methylphenyl                                                                           --CSNH--                                                                             F  H                                              64    5a  4-methylphenyl                                                                           --     -- Et                                             65    5a  4-methylphenyl                                                                           --     -- H                                              66    2   2-furyl    --C.tbd.C--                                                                          H  Et                                             67    2   2-furyl    --C.tbd.C--                                                                          H  H                                              __________________________________________________________________________

Aryl and (3-Oxo-1-Propenyl)-Substituted Benzopyran, Benzothiopyran,Dihydroquinoline and 5,6-Dihydronaphthalene Derivatives Having RetinoidAntagonist-Like Biological Activity

With reference to the symbol Y in Formula 101, the preferred compoundsof the invention are those where Y is phenyl, pyridyl, thienyl or furyl.Even more preferred are compounds where Y is phenyl or pyridyl, andstill more preferred where Y is phenyl. As far as substitutions on the Y(phenyl) and Y (pyridyl) groups are concerned, compounds are preferredwhere the phenyl group is 1,4 (para) substituted by the --CR₁₆ ═CR₁₇ --and A-B groups, and where the pyridine ring is 2,5 substituted by the--CR₁₆ ═CR₁₇ -- and A-B groups. (Substitution in the 2,5 positions inthe "pyridine" nomenclature corresponds to substitution in the 6-position in the "nicotinic acid" nomenclature.) In the preferredcompounds of the invention there is no optional R₂ substituent on the Ygroup.

The A-B group of the preferred compounds is (CH₂)_(n) --COOH or(CH₂)_(n) --COOR₈, where n and R₈ are defined as above. Even morepreferably n is zero and R₈ is lower alkyl, or n is zero and B is COOHor a pharmaceutically acceptable salt thereof.

In the presently preferred examples of compounds of the invention X isC(R₁)₂ ! where n is 1. Nevertheless, compounds where X is S or O(benzothiopyran and benzopyran derivatives) are also preferred. When Xis C(R₁)₂ !_(n) and n is 1, then R₁ preferably is alkyl of 1 to 6carbons, even more preferably methyl.

The R₂ group attached to the aromatic portion of thetetrahydronaphthalene, benzopyran, benzothiopyran or dihydroquinolinemoiety of the compounds of Formula 101 is preferably H, F or CF₃. R₃ ispreferably hydrogen or methyl, even more preferably hydrogen.

Referring now to the R₁₄ group, compounds are preferred where R₁₄ isphenyl, 2-pyridyl, 3-pyridyl, 2- thienyl, and 2-thiazolyl. The R₁₅ group(substituent of the R₁₄ group) is preferably H, lower alkyl,trifluoromethyl, chlorine, lower alkoxy or hydroxy.

Preferred compounds of the invention are shown in Table 2 with referenceto Formula 102. ##STR4##

                  TABLE 2                                                         ______________________________________                                        Compund          R.sub.15 *                                                                            R.sub.8 *                                            ______________________________________                                        101              CH.sub.3                                                                              H                                                    102              CH.sub.3                                                                              Et                                                   103              H       H                                                    104              H       Et                                                   ______________________________________                                    

Biological Activity, Modes of Administration

As noted above, the compounds of the present invention are antagonistsof one or more RAR receptor subtypes. This means that the compounds ofthe invention bind to one or more RAR receptor subtypes, but do nottrigger the response which is triggered by agonists of the samereceptors. Some of the compounds of the present invention areantagonists of all three RAR receptor subtypes (RAR-α, RAR-β and RAR-γ),and these are termed "RAR pan antagonists". Some others are antagonistsof only one or two of RAR receptor subtypes. Some compounds within thescope of the present invention are partial agonists of one or two RARreceptor subtypes and antagonists of the remaining subtypes. Thecompounds of the invention do not bind to RXR receptors, therefore theyare neither agonists nor antagonists of RXR.

Depending on the site and nature of undesirable side effects which aredesired to be suppressed or ameliorated, compounds used in accordancewith the invention may be antagonists of only one or two of RAR receptorsubtypes. Some compounds used in accordance with the invention may bepartial agonists of one or two RAR receptor subtypes and antagonists ofthe remaining subtypes. Such compounds are, generally speaking, usablein accordance with the invention if the antagonist effect is on that RARreceptor subtype (or subtypes) which is (are) predominantly responsiblefor the overdose poisoning or for the undesired side effect or sideeffects. In this connection it is noted that, generally speaking, acompound is considered an antagonist of a given receptor subtype if inthe below described co-tranfection assays the compound does not causesignificant transcriptional activation of the receptor regulatedreporter gene, but nevertheless binds to the receptor with a K_(d) valueof less than approximately 1 μM.

Whether a compound is an RAR antagonist and therefore can be utilized inaccordance with the present invention, can be tested in the followingassays.

A chimeric receptor transactivation assay which tests for agonist-likeactivity in the RAR-α, RAR-β, RAR-γ, RXR-α receptor subtypes, and whichis based on work published by Feigner P. L. and Holm M. Focus Vol 11,No. 2 (1989) is described in detail in published PCT Application No.W094/17796, published on Aug. 18, 1994. The latter publication is thePCT counterpart of U. S. application Ser. No. 08/016,404, filed on Feb.11, 1993, which issued as U.S. Pat. No. 5,455,265. PCT publicationW094/17796 and the specification of U.S. Pat. No. 5,455,265 are herebyexpressly incorporated by reference. A compound should not causesignificant activation of a reporter gene through a given receptorsubtype (RAR-α, RAR-β or RAR-γ) in this assay, in order to qualify as anRAR antagonist with utility in the present invention.

A holoreceptor transactivation assay and a ligand binding assay whichmeasure the antagonist/agonist like activity of the compounds of theinvention, or their ability to bind to the several retinoid receptorsubtypes, respectively, are described in published PCT Application No.W093/11755 (particularly on pages 30-33 and 37-41) published on Jun. 24,1993, the specification of which is also incorporated herein byreference. A description of the holoreceptor transactivation assay isalso provided below.

Holoreceptor Transactivation Assay

CV1 cells (5,000 cells/well) were transfected with an RAR reporterplasmid MTV-TREp-LUC (50 ng) along with one of the RAR expressionvectors (10 ng) in an automated 96-well format by the calcium phosphateprocedure of Heyman et al. Cell 68: 397-406. For RXR-α and RXR-γtransactivation assays, an RXR-responsive reporter plasmid CRBPII-tk-LUC(50 ng) along with the appropriate RXR expression vectors (10 ng) wasused substantially as described by Heyman et al. above, and Allegrettoet al. J Biol. Chem. 268: 26625-26633. For RXR-β transactivation assays,an RXR-responsive reporter plasmid CPRE-tk-LUC (50 mg) along with RXR-βexpression vector (10 mg) was used as described in above. Thesereporters contain DRI elements from human CRBPII and certain DRIelements from promotor, respectively (see Mangelsdorf et al. TheRetinoids: Biology Chemistry and Medicine, pp. 319-349, Raven PressLtd., New York and Heyman et al., cited above). A β-galactosidase (50ng) expression vector was used as an internal control in thetransfections to normalize for variations in transfection efficiency.The cells were transfected in triplicate for 6 hours, followed byincubation with retinoids for 36 hours, and the extracts were assayedfor luciferase and β-galactosidase activities. The detailed experimentalprocedure for holoreceptor transactivations has been described in Heymanet al. above, and Allegretto et al. cited above. The results obtained inthis assay are expressed in EC₅₀ numbers, as they are also in thechimeric receptor transactivation assay. The Heyman et al. Cell 68:397-406, Allegretto et al. J Biol. Chem. 268: 26625-26633, andMangelsdorf et al. The Retinoids: Biology Chemistry and Medicine, pp.319-349, Raven Press Ltd., New York, are expressly incorporated hereinby reference. The results of ligand binding assay are expressed in K_(d)numbers. (See Cheng et al. Biochemical Pharmacology 22: 3099-3108,expressly incorporated herein by reference.)

A compound should not cause significant activation of a reporter genethrough a given receptor subtype (RAR-α, RAR-β or RAR-γ) in theholoreceptor transactivation assay assay, in order to qualify as an RARantagonist with utility in the present invention. Last, but not least, acompound should bind to at least one of the RAR receptor subtypes in theligand binding assay with a K_(d) of less than approximately 1micromolar (K_(d) <1 μM) in order to be capable of functioning as anantagonist of the bound receptor subtype, provided the same receptorsubtype is not significantly activated by the compound.

Table 3 below shows the results of the holoreceptor transactivationassay and Table 4 discloses the efficacy (in percentage) in this assayof the test compound relative to all trans retinoic acid, for certainexemplary compounds of the invention. Table 5 shows the results of theligand binding assay for certain exemplary compounds of the invention.

                  TABLE 3                                                         ______________________________________                                        Holoreceptor Transactivation Assay                                            Compound EC.sub.50 (nanomolar)                                                #        RARα                                                                            RARβ                                                                             RARγ                                                                          RXRα                                                                          RXRβ                                                                           RXRγ                         ______________________________________                                        18       0.00    0.00    0.00  0.00  0.00  0.00                               19       0.00    0.00    0.00  0.00  0.00  0.00                               20       0.00    0.00    0.00  0.00  0.00  0.00                               21       0.00    0.00    0.00  0.00  0.00  0.00                               22       0.00    0.00    0.00  0.00  0.00  0.00                               23       0.00    0.00    0.00  0.00  0.00  0.00                               24       0.00    0.00    0.00  0.00  0.00  0.00                               25       0.00    0.00    0.00  0.00  0.00  0.00                               26       0.00    0.00    0.00  0.00  0.00  0.00                               27       0.00    0.00    0.00  0.00  0.00  0.00                               28       0.00    0.00    0.00  0.00  0.00  0.00                               29       0.00    0.00    0.00  0.00  0.00  0.00                               30       0.00    0.00    0.00  0.00  0.00  0.00                               31       0.00    0.00    0.00  0.00  0.00  0.00                               32       0.00    0.00    0.00  0.00  0.00  0.00                               34       0.00    0.00    0.00  0.00  0.00  0.00                               36       0.00    0.00    0.00  0.00  0.00  0.00                               39       0.00    0.00    0.00  0.00  0.00  0.00                               41       0.00    0.00    0.00  0.00  0.00  0.00                               45       0.00    0.00    0.00  0.00  0.00  0.00                               46b      0.00    0.00    0.00  0.00  0.00  0.00                               52       0.00    0.00    0.00  0.00  0.00  0.00                               60       0.00    0.00    0.00  0.00  0.00  0.00                               61       0.00    0.00    0.00  0.00  0.00  0.00                               63       0.00    0.00    0.00  0.00  0.00  0.00                               101      0.00    0.00    0.00  0.00  0.00  0.00                               103      0.00    0.00    0.00  0.00  0.00  0.00                               ______________________________________                                         0.0 in Table 3 indicates that the compound is less than 20% as active         (efficacious) in this assay than all trans retinoic acid.                

                  TABLE 4                                                         ______________________________________                                        Transactivation Assay Efficacy (% of RA activity)                             Compound                                                                      #        RARα                                                                            RARβ                                                                             RARγ                                                                          RXRα                                                                          RXRβ                                                                           RXRγ                         ______________________________________                                        18       4.00    1.00    0.00  2.00  10.00 1.0                                19       0.00    5.0     3.0   0.0   9.0   4.0                                20       3.0     4.0     0.00  4.00  0.00  3.0                                21       2.00    2.00    2.00  3.00  0.00  3.00                               22       0.00    0.00    2.00  1.00  0.00  2.00                               23       0.00    6.00    3.00  1.00  0.00  4.00                               24       3.00    7.00    4.00  1.00  0.00  3.00                               25       2.00    3.00    3.00  5.00  0.00  3.00                               26       1.00    6.00    0.00  2.00  0.00  3.00                               27       9.00    14.00   6.00  2.00  0.00  4.00                               28       2.00    10.00   2.00  2.00  0.00  3.00                               29       0.00    6.00    11.00 0.00  6.00  2.00                               30       3.00    5.00    1.00  0.00  9.00  3.00                               31       4.00    14.00   2.00  1.00  8.00  6.00                               32       0.00    2.00    2.00  1.00  0.00  2.00                               34       3.00    5.00    2.00  1.00  0.00  3.00                               36       1.00    5.00    0.00  1.00  7.00  2.00                               39       1.00    7.00    9.00  2.00  0.00  1.00                               41       3.00    5.00    6.00  1.00  0.00  3.00                               45       2.00    0.00    7.00  3.00  8.00  0.00                               46b      4.00    5.00    3.00  2.00  0.00  4.00                               52       0.00    15.00   3.00  0.00  0.00  10.00                              60       0.00    1.00    4.00  3.00  0.00  3.00                               61       2.00    2.00    0.00  1.00  0.00  3.00                               63       2.00    2.00    7.00  1.00  0.00  1.00                               101      0.00    4.00    2.00  1.00  0.00  3.0                                103      4.00    12.0    7.0   0.00  0.0   2.0                                ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Ligand Binding Assay                                                          Compound K.sub.d (nanomolar)                                                  #        RARα                                                                            RARβ                                                                             RARγ                                                                          RXRα                                                                          RXRβ                                                                           RXRγ                         ______________________________________                                        18       24.00   11.00   24.00 0.00  0.00  0.00                               19       565     210     659   0.00  0.00  0.00                               20       130.00  22.0    34.00 0.00  0.00  0.00                               21       16      9       13    0.00  0.00  0.00                               22       24.0    17.0    27.0  0.00  0.00  0.00                               23       32.00   25.00   31.00 0.00  0.00  0.00                               24       699     235     286   0.00  0.00  0.00                               25       50      17      20    0.00  0.00  0.00                               26       40.00   31.00   36.00 0.00  0.00  0.00                               27       69.00   14.00   26.00 0.00  0.00  0.00                               28       669     77      236   0.00  0.00  0.00                               29       234     48      80    0.00  0.00  0.00                               30       683     141     219   0.00  0.00  0.00                               31       370     52.00   100.00                                                                              0.00  0.00  0.00                               32       0.00    89.00   169.00                                                                              0.00  0.00  0.00                               34       52.00   30.00   17.00 0.00  0.00  0.00                               36       13.00   550.00  0.00  0.00  0.00  0.00                               39       67.00   38.00   113.00                                                                              0.00  0.00  0.00                               41       5.1     491     725   0.00  0.00  0.00                               45       12.0    2.80    17.0  0.00  0.00  0.00                               46b      250     3.70    5.80  0.00  0.00  0.00                               52       60.00   63.00   56.00 0.00  0.00  0.00                               60       1.5     1.9     3.3   0.00  0.00  0.00                               61       96      15      16    0.00  0.00  0.00                               63       133     3219    0.00  0.00  0.00  0.00                               101      750     143     637   0.00  0.00  0.00                               103      301     273     261   0.00  0.00  0.00                               ______________________________________                                         0.0 in Table 5 indicates a value greater than 1000 nM.                   

As it can be seen from the test results summarized in Tables 3, 4 and 5,the therein indicated exemplary compounds of the invention areantagonists of the RAR receptor subtypes, but have no affinity to RXRreceptor subtypes. (Other compounds of the invention may be antagonistof some but not all RAR receptor subtypes and agonists of the remainingRAR subtypes.) Due to this property, the compounds of the invention canbe used to block the activity of RAR agonists in biological assays. Inmammals, including humans, the compounds of the invention can becoadministered with RAR agonists and, by means of pharmacologicalselectivity or site-specific delivery, preferentially prevent theundesired effects of RAR agonists. The compounds of the invention canalso be used to treat Vitamin A overdose, acute or chronic, resultingeither from the excessive intake of vitamin A supplements or from theingestion of liver of certain fish and animals that contain high levelof Vitamin A. Still further, the compounds of the invention can also beused to treat acute or chronic toxicity caused by retinoid drugs. It hasbeen known in the art that the toxicities observed with hypervitaminosisA syndrome (headache, skin peeling, bone toxicity, dyslipidemias) aresimilar or identical with toxicities observed with other retinoids,suggesting a common biological cause, that is RAR activation. Becausethe compounds of the present invention block RAR activation, they aresuitable for treating the foregoing toxicities.

The compounds of the invention are able to substantially prevent skinirritation induced by RAR agonist retinoids, when the compound of theinvention is topically coadministered to the skin. Similarly, compoundsof the invention can be administered topically to the skin, to blockskin irritation, in patients or animals who are administered RAR agonistcompounds systemically. The compounds of the invention can acceleraterecovery from pre-existing retinoid toxicity, can blockhypertriglyceridemia caused by co- administered retinoids, and can blockbone toxicity induced by an RAR agonist (retinoid).

Generally speaking, for therapeutic applications in mammals inaccordance with the present invention, the antagonist compounds can beadmistered enterally or topically as an antidote to vitamin A, vitamin Aprecursors, or antidote to retinoid toxicity resulting from overdose orprolonged exposure, after intake of the causative factor (vitamin Aprecursor or other retinoid) has been discontinued. Alternatively, theantagonist compounds are coadministered with retinoid drugs inaccordance with the invention, in situations where the retinoid providesa therapeutic benefit, and where the coadministered antagonistalleviates or eliminates one or more undesired side effects of theretinoid. For this type of application the antagonist may beadministered in a site-specific manner, for example as a topicallyapplied cream or lotion while the coadministered retinoid may be givenenterally.

For therapeutic applications in accordance with the present inventionthe antagonist compounds are incorporated into pharmaceuticalcompositions, such as tablets, pills, capsules, solutions, suspensions,creams, ointments, gels, salves, lotions and the like, using suchpharmaceutically acceptable excipients and vehicles which per se arewell known in the art. For example preparation of topical formulationsare well described in Remington's Pharmaceutical Science, Edition 17,Mack Publishing Company, Easton, Pa. For topical application, theantagonist compounds could also be administered as a powder or spray,particularly in aerosol form. If the drug is to be administeredsystemically, it may be confected as a powder, pill, tablet or the likeor as a syrup or elixir suitable for oral administration. Forintravenous or intraperitoneal administration, the antagonist compoundwill be prepared as a solution or suspension capable of beingadministered by injection. In certain cases, it may be useful toformulate the antagonist compounds by injection. In certain cases, itmay be useful to formulate the antagonist compounds in suppository formor as extended release formulation for deposit under the skin orintramuscular injection.

The antagonist compounds will be administered in a therapeuticallyeffective dose in accordance with the invention. A therapeuticconcentration will be that concentration which effects reduction of theparticular condition (such as toxicity due to retinoid or vitamin Aexposure, or side effect of retinoid drug) or retards its expansion. Itshould be understood that when coadministering the antagonist compoundsto block retinoid-induced toxicity or side effects in accordance withthe invention, the antagonist compounds are used in a prophylacticmanner to prevent onset of a particular condition, such as skinirritation.

A useful therapeutic or prophylactic concentration will vary fromcondition to condition and in certain instances may vary with theseverity of the condition being treated and the patient's susceptibilityto treatment. Accordingly, no single concentration will be uniformlyuseful, but will require modification depending on the particularitiesof the chronic or acute retinoid toxicity or related condition beingtreated. Such concentrations can be arrived at through routineexperimentation. However, it is anticipated that a formulationcontaining between 0.01 and 1.0 milligrams of antagonist compound permililiter of formulation will constitute a therapeutically effectiveconcentration for topical application. If administered systemically, anamount between 0.01 and 5 mg per kg per day of body weight would beexpected to effect a therapeutic result.

The basis of the utility of RAR antagonists for the prevention ortreatment of RAR agonist-induced toxicity is competitive inhibition ofthe activation of RAR receptors by RAR agonists. The main distinctionbetween these two applications of RAR antagonists is the presence orabsence of preexisting retinoid toxicity. Most of the examplesimmediately described below relate to the use of retinoids to preventretinoid toxicity, but the general methods described herein areapplicable to the treatment of preexisting retinoid toxicity as well.

Description of experiments demonstrating the use of RAR antagonists toprevent or treat retinoid toxicity and/or side effects of retinoid drugsEXAMPLE 1: skin iritation induced by topically applied agonist istreated with topically applied antagonist

The compound 4- (E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl)propen-1-yl!benzoic acid, designated AGN191183, is known in the prior art as a potent RAR agonist (see forexample the descriptive portion and FIG. 2b of U.S. Pat. No. 5,324,840).(The "AGN" number is an arbitrarily designated reference number utilizedby the corporate assignee of the present invention for identification ofcompounds.)

4- (5,6-dihydro-5,5-dimethyl-8-(phenyl)-2-naphthalenyl)ethynyl!benzoicacid (AGN 192869, also designated Compound 60a) is a compound thepreparation of which is described below. This compound is an RARantagonist.

Skin irritation induced by an RAR agonist, AGN 191183, administeredtopically, can be blocked by an RAR antagonist, AGN 192869, alsoadministered topically in hairless mice.

More particularly skin irritation was measured on a semiquantitativescale by the daily subjective evaluation of skin flaking and abrasions.A single number, the topical irritation score, summarizes the skinirritation induced in an animal during the course of an experiment. Thetopical irritation score is calculated as follows. The topicalirritation score is the algebraic sum of a composite flaking score and acomposite abrasion score. The composite scores range from 0-9 and 0-8for flaking and abrasions, respectively, and take into account themaximum severity, the time of onset, and the average severity of theflaking and abrasions observed.

The severity of flaking is scored on a 5-point scale and the severity ofabrasions is scored on a 4- point scale, with higher scores reflectinggreater severity. The maximum severity component of the composite scoreswould be the highest daily severity score assigned to a given animalduring the course of observation.

For the time of onset component of the composite score, a score rangingfrom 0 to 4 is assigned as follows:

                  TABLE 6                                                         ______________________________________                                        Time to Appearance of Flaking or Abrasions                                    od Severity 2 or Greater                                                      (days)      Time of Onset Score                                               ______________________________________                                        8           0                                                                 6-7         1                                                                 5           2                                                                 3-4         3                                                                 1-2         4                                                                 ______________________________________                                    

The average severity component of the composite score is the sum of thedaily flaking or abrasion scores divided by the number of observationdays. The first day of treatment is not counted, since the drug compoundhas not had an opportunity to take effect at the time of firsttreatment.

To calculate the composite flaking and abrasion scores, the averageseverity and time of onset scores are summed and divided by 2. Theresult is added to the maximal severity score. The composite flaking andabrasion scores are then summed to give the overall topical irritationscore. Each animal receives a topical irritation score, and the valuesare expressed as the mean ±SD of the individual scores of a group ofanimals. Values are rounded to the nearest integer.

Female hairless mice Cr1:SKH1-hrBR! (8-12 weeks old, n=6) were treatedtopically for 5 consecutive days with acetone, AGN 191183, AGN 192869,or some combination of AGN 192869 and 191183. Doses of the respectivecompounds are given in Table 7. Treatments are applied to the dorsalskin in a total volume of 4 ml/kg (˜0.1 ml). Mice were observed dailyand scored for flaking and abrasions up to and including 3 days afterthe last treatment, i.e., day 8.

                  TABLE 7                                                         ______________________________________                                        Experimental Design and Results, Example 1                                          Dose        Dose       Molar Ratio                                                                            Topical                                       AGN 191183  AGN 192869 (192869: Irritation                              Group (mg/kg/d)   (mg/kg/d)  (191183  Score)                                  ______________________________________                                        A     0           0          --       0 ± 0                                B     0.025       0          --       8 ± 2                                C     0.025       0.06        2:1     5 ± 2                                D     0.025       0.30       10:1     2 ± 1                                E     0.025       1.5        50:1     1 ± 0                                F     0           1.5        --       0 ± 0                                ______________________________________                                    

The topical irritation scores for Example 1 are given in Table 7.Neither acetone (vehicle) nor AGN 192869 (antagonist) at a dose of 1.5mg/kg/d (group F) caused observable topical irritation. AGN 191183, theRAR agonist, caused modest topical irritation at a dose of 0.025mg/kg/d. However, AGN 191183-induced topical irritation was inhibited ina dose-dependent fashion by AGN 192869, with nearly complete abrogationof irritation in the presence of a 50-fold molar excess of AGN 192869.This demonstrates that a topical RAR antagonist blocks skin irritationcaused by a topical RAR agonist. Complete blockade of RARagonist-induced skin irritation can be achieved with lower molar ratiosof antagonist to agonist when the RAR antagonists is more potent, suchas the compound 4- (5,6-dihydro-5,5- dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoic acid (AGN 193109, also designated in thisapplication as Compound 60.)

EXAMPLE 2: skin iritation induced by orally applied agonist is blockedwith topically applied antagonist

The potent RAR agonist AGN 191183 (4-(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl)propen-1-yl!benzoicacid) and the potent RAR antagonist 4- (5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoic acid (AGN 193109, Compound60) were used in this example and body weight of the experimentalanimals (mice) was used as a marker of systemic RAR agonist exposure.

Groups of female hairless mice (8-12 weeks old, n=6) were treated byintragastric intubation with corn oil or AGN 191183 (0.26 mg/kg)suspended in corn oil (5 ml/kg). Mice were simultaneously treatedtopically on the dorsal skin with vehicle (97.6% acetone/2.4%dimethylsulfoxide) or solutions of AGN 193109 in vehicle (6 ml/kg).Specific doses for the different treatment groups are give in Table 8.Treatments were administered daily for 4 consecutive days. Mice wereweighed and graded for topical irritation daily as described in Example1 up to and including 1 day after the last treatment. Percent bodyweight change is calculated by subtracting final body weight (day 5)from initial body weight (day 1), dividing by initial body weight, andmultiplying by 100%. Topical irritation scores are calculated asdescribed in Example 1.

Topical irritation scores and weight loss for the different groups aregiven in Table 8. Combined treatment with the topical and oral vehicles,i.e., acetone and corn oil, respectively, caused no topical irritationor weight loss. Similarly, combined treatment with the oral vehicle andthe topical antagonist AGN 193109 resulted in no topical irritation orweight loss. Oral AGN 191183 by itself induced substantial weight lossand skin irritation. AGN 191183-induced skin irritation wassubstantially reduced when combined with the lower dose of AGN 193109and completely blocked at the higher dose of AGN 193109. AGN191183-induced weight loss was also blocked in a dose-related fashion bytopical AGN 193109, but the blockade was not complete. Thus, topical AGN193109 preferentially blocked the dermal toxicity of AGN 191183.Presumably, low levels of AGN 193109 were absorbed systemically and thuspartially blocked the weight loss induced by AGN 191183. However, suchabsorption would likely be even less in a species with less permeableskin, such as humans. Alternatively, the weight loss inhibition by AGN193109 could be due to amelioration of the AGN 191183 induced skinirritation.

                  TABLE 8                                                         ______________________________________                                        Experimental Design and Results, Example 2                                           Dose of Topical                                                                           Dose od Oral                                                                             % Weight                                                                             Topical                                         AGN 193109  AGN 191183 Gain or                                                                              Irritation                               Group  (mg/kg/d)   (mg/kg/d)  (Loss) Score)                                   ______________________________________                                        A      0           0          1 ± 2                                                                             0 ± 0                                 B      0           0.26       (21 ± 6)                                                                          8 ± 1                                 C      0.12        0.26       (9 ± 5)                                                                           1 ± 1                                 D      0.47        0.26       (3 ± 5                                                                            0 ± 1                                 E      0.47        0          3 ± 3                                                                             0 ± 0                                 ______________________________________                                    

Thus, Example 2 demonstrates that RAR antagonists administered topicallycan be used to block preferentially the skin irritation induced by anRAR agonist administered orally.

EXAMPLE 3: topically applied antagonist accelarates recovery fromprexisting retinoid toxicity

In this example, weight loss is induced by topical treatment with theRAR agonist AGN 191183 and then the test animals are topically treatedwith either vehicle or the RAR antagonist AGN 193109.

Female hairless mice (8-12 weeks old, n=5) were treated topically withAGN 191183 (0.13 mg/kg/d) in vehicle (97.6% acetone/2.4% DMSO, 4 ml/kg)daily for 2 days. Groups of these same mice (n=5) were then treatedtopically either with vehicle or AGN 193109 in vehicle (4 ml/kg) dailyfor 3 consecutive days beginning on day 3. Mice were weighed on days 1-5and on day 8. Body weights are expressed as the mean ±SD. Means werecompared statistically using an unpaired, two-tailed t-test. Differenceswere considered significant at P<0.05.

                                      TABLE 9                                     __________________________________________________________________________    Results, Example 3                                                            Treatment                                                                            Body Weight (g)                                                        (days 3-5)                                                                           DAY 1                                                                              DAY 2                                                                              DAY 3                                                                              DAY 4                                                                              DAY 5                                                                              DAY 8                                         __________________________________________________________________________    vehicle                                                                              24.6 ± 1.5                                                                      23.9 ± 1.2                                                                      21.4 ± 1.2                                                                      20.3 ± 1.7                                                                      21.0 ± 1.4                                                                      24.7 ± 1.0                                 AGN 193109                                                                           23.9 ± 1.0                                                                      23.5 ± 1.2                                                                      21.4 ± 0.6                                                                      22.2 ± 0.7                                                                      22.8 ± 0.8                                                                      25.0 ± 1.1                                 __________________________________________________________________________

The time course of body weights in Example 3 are given in Table 9. Bodyweights of both groups of mice were lowered in parallel on days 2 and 3as a result of AGN 191183 treatment on days 1 and 2. Body weights in thetwo groups were not significantly different on days 1, 2, or 3. However,AGN 193109 treatment significantly increased body weight relative tovehicle treatment on days 4 and 5. These data indicated that recoveryfrom AGN 191183-induced body weight loss was accelerated by subsequenttreatment with AGN 193109. Body weights were not significantly differentbetween the two groups of mice on day 8, indicating that full recoverywas achievable in both groups given sufficient time. Thus, RARantagonists are effective in alleviating RAR agonist-induced toxicityeven if RAR agonist-induced toxicity precedes RAR antagonist treatment,i.e., in the RAR agonist poisoning scenario.

EXAMPLE 4: orally applied antagonist blocks hypertriglyceridemia incudedby orally coadministered retinoid agonist

5-(E)-2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethylnaphthalen-2-yl)propen-1-yl!-2-thiophencarboxylicacid, is a known RAR/RXR pan- agonist (see U.S. Pat. No. 5,324,840column 32) and is designated AGN 191659. This compound was used orallyto induce acute hypertriglyceridemia in rats, and AGN 193109 Compound 60was coadministered orally to block the AGN 191659-inducedhypertriglyceridemia.

Male Fischer rats (6-7 weeks old, n=5) were treated by intragastricintubation with corn oil (vehicle), AGN 191659, AGN 193109 or acombination of AGN 191659 and AGN 193109. AGN 191659 and AGN 193109 weregiven as fine suspensions in corn oil. The experimental design,including doses, is given in Table 10.

Blood was withdrawn from the inferior vena cava under carbon dioxidenarcosis. Serum was separated from blood by low speed centrifugation.Total serum triglycerides (triglycerides plus glycerol) were measuredwith a standard spectrophotometric endpoint assay available commerciallyas a kit and adapted to a 96-well plate format. Serum triglyceridelevels are expressed as the mean ±SD. Means were compared statisticallyby one-way analysis of variance followed by Dunnett's test ifsignificant differences were found. Differences were consideredsignificant at P<0.05.

As shown in Table 10, AGN 191659 by itself caused significant elevationof serum triglycerides relative to vehicle treatment. AGN 193109 byitself did not significantly increase serum triglycerides. Importantly,the combination of AGN 193109 and AGN 191659 at molar ratios of 1:1 and5:1 reduced serum triglycerides to levels that were not signficantlydifferent from control.

                  TABLE 10                                                        ______________________________________                                        Experimental Design and Results, Example 4                                    Group  Treatment (dose) Serum Triglycerides (mg/dl)                           ______________________________________                                        A      vehicle          55.0 ± 3.1                                         B      AGN 193109 (19.6 mg/kg)                                                                        52.4 ± 6.3                                         C      AGN 191659 (3.7 mg/kg)                                                                         122.5 ± 27.6                                       D      AGN 193109 (3.9 mg/kg) +                                                                        55.7 ± 14.7                                              AGN 191659 (3.7 mg/kg)                                                 E      AGN 193109 (19.6 mg/kg) +                                                                      72.7 ± 8.9                                                AGN 191659 (3.7 mg/kg)                                                 ______________________________________                                    

Example 4 demonstrates that an RAR antagonist can be used to blockhypertriglyceridemia induced by a coadministered retinoid.

EXAMPLE 5: parenterally applied antagonist blocks bone toxicity incudedby parenterally coadministered retinoid agonist

Example 5 demonstrates that RAR antagonists can block bone toxicityinduced by an RAR agonist. In this example, AGN 193109 is used to blockpremature epiphyseal plate closure caused by a coadministered RARagonist, AGN 191183, in guinea pigs.

Groups of male Hartley guinea pigs (˜3 weeks old, n=4) were implantedintraperitoneally with osmotic pumps containing vehicle (20%dimethylsulfoxide/80% polyethylene glycol-300), AGN 191183 (0.06 mg/ml),or AGN 191183 (0.06 mg/ml) in combination with AGN 193109 (0.34 mg/ml).The osmotic pumps are designed by the manufacturer to deliver ˜5 μl ofsolution per hour continuously for 14 days.

The animals were euthanized by carbon dioxide asphyxiation 14 days afterimplantation. The left tibia was was removed and placed in 10% bufferedformalin. The tibias were decalcified by exposure to a formicacid/formalin solution for 3-4 days, and paraffin sections wereprepared. Sections were stained with hematoxylin and eosin by standardmethods. The proximal tibial epiphyseal plate was examined and scored asclosed or not closed. Epiphyseal plate closure is defined for thispurpose as any interruption of the continuity of the epiphyseal growthplate cartilage, i.e., replacement by bone and/or fibroblastic tissue.

None of the four vehicle-treated guinea pigs showed epiphyseal plateclosure by the end of the experiment. This was expected, since theproximal epiphyseal plate of guinea pig tibia does not normally closeuntil the animals are at least 10 months old. All four of the AGN191183-treated guinea pigs showed partial or complete epiphyseal plateclosure. However, none of the guinea pigs treated with the combinationof AGN 191183 and AGN 193109 demonstrated epiphyseal plate closure.Thus, AGN 193109 at a 5-fold molar excess completely blocked AGN191183-induced bone toxicity when these compounds were coadministeredparenterally.

RAR Antagonist Compounds

The compounds 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoicacid (AGN 193109, Compound 60) and 4-(5,6-dihydro-5,5-dimethyl-8-(phenyl)-2-naphthalenyl)ethynyl!benzoic acid(AGN 192869, Compound 60a) are examples of RAR antagonists which wereused in the above-described animal tests for blocking RAR receptors inaccordance with the present invention. The compounds of the followingformula (Formula 1) serve as further and general examples for additionalRAR antagonist compounds for use in accordance with the presentinvention. ##STR5##

In Formula 1, X is S, O, NR' where R' is H or alkyl of 1 to 6 carbons,or

X is C(R₁)₂ !_(n) where R₁ is H or alkyl of 1 to 6 carbons, and n is aninteger between 0 or 1;

R₂ is hydrogen, lower alkyl of 1 to 6 carbons, F, CF₃, fluor substitutedalkyl of 1 to 6 carbons, OH, SH, alkoxy of 1 to 6 carbons, or alkylthioof 1 to 6 carbons;

R₃ is hydrogen, lower alkyl of 1 to 6 carbons or F;

m is an integer having the value of 0-3;

o is an integer having the value of 0-3;

Z is --C═C--,

--N═N--,

--N═CR₁ --,

--CR₁ ═N,

--(CR₁ ═CR₁)_(n) -- where n' is an integer having the value 0-5,

--CO--NR₁ --,

--CS--NR₁ --,

--NR₁ --CO,

--NR₁ --CS,

--COO--,

--OCO--;

--CSO--;

--OCS--;

Y is a phenyl or naphthyl group, or heteroaryl selected from a groupconsisting of pyridyl, thienyl, furyl, pyridazinyl, pyrimidinyl,pyrazinyl, thiazolyl, oxazolyl, imidazolyl and pyrrazolyl, said phenyland heteroaryl groups being optionally substituted with one or two R₂groups, or

when Z is --(CR₁ ═CR₁)_(n) -- and n' is 3, 4 or 5 then Y represents adirect valence bond between said (CR₂ ═CR₂)_(n), group and B;

A is (CH₂)_(q) where q is 0-5, lower branched chain alkyl having 3-6carbons, cycloalkyl having 3-6 carbons, alkenyl having 2-6 carbons and 1or 2 double bonds, alkynyl having 2-6 carbons and 1 or 2 triple bonds;

B is hydrogen, COOH or a pharmaceutically acceptable salt thereof,COOR₈, CONR₉ R₁₀, --CH₂ OH, CH₂ OR₁₁, CH₂ OCOR₁₁, CHO, CH(OR₁₂)₂, CHOR₁₃O, --COR₇, CR₇ (OR₁₂)₂, CR₇ OR₁₃ O, or tri-lower alkylsilyl, where R₇ isan alkyl, cycloalkyl or alkenyl group containing 1 to 5 carbons, R₈ isan alkyl group of 1 to 10 carbons or trimethylsilylalkyl where the alkylgroup has 1 to 10 carbons, or a cycloalkyl group of 5 to 10 carbons, orR₈ is phenyl or lower alkylphenyl, R₉ and R₁₀ independently arehydrogen, an alkyl group of 1 to 10 carbons, or a cycloalkyl group of5-10 carbons, or phenyl or lower alkylphenyl, R₁₁ is lower alkyl, phenylor lower alkylphenyl, R₁₂ is lower alkyl, and R₁₃ is divalent alkylradical of 2-5 carbons, and

R₁₄ is (R₁₅)_(r) -phenyl, (R₁₅)_(r) -naphthyl, or (R₁₅)_(r) --heteroaryl where the heteroaryl group has 1 to 3 heteroatoms selectedfrom the group consisting of O, S and N, r is an integer having thevalues of 0-5, and

R₁₅ is independently H, F, Cl, Br, I, NO₂, N(R₈)₂, N(R₈)COR₈, NR₈CON(R₈)₂, OH, OCOR₈, OR₈, CN, an alkyl group having 1 to 10 carbons,fluoro substituted alkyl group having 1 to 10 carbons, an alkenyl grouphaving 1 to 10 carbons and 1 to 3 double bonds, alkynyl group having 1to 10 carbons and 1 to 3 triple bonds, or a trialkylsilyl ortrialkylsilyloxy group where the alkyl groups independently have 1 to 6carbons.

Synthetic Methods--Aryl Substituted Compounds

The exemplary RAR antagonist compounds of Formula 1 can be made by thesynthetic chemical pathways illustrated here. The synthetic chemist willreadily appreciate that the conditions set out here are specificembodiments which can be generalized to any and all of the compoundsrepresented by Formula 1. ##STR6##

Reaction Scheme 1 illustrates the synthesis of compounds of Formula 1where the Z group is an ethynyl function (--C.tbd.C--) and X is C(R₁)₂!_(n) where n is 1. In other words, Reaction Scheme 1 illustrates thesynthesis of ethynyl substituted dihydronaphthalene derivatives of thepresent invention. In accordance with this scheme, atetrahydronaphtalene-1-one compound of Formula 6 is brominated toprovide the bromo derivative of Formula 7. The compounds of Formula 6already carry the desired R₁, R₂ and R₃ substituents, as these aredefined above in connection with Formula 1. A preferred example of acompound of Formula 6 is 3,4-dihydro-4,4-dimethyl-1(2H)-- naphthalenone,which is described in the chemical literature (Arnold et al. J Am. Chem.Soc. 69: 2322-2325 (1947)). A presently preferred route for thesynthesis of this compound from 1-bromo-3-phenylpropane is alsodescribed in the experimental section of the present application.

The compounds of Formula 7 are then reacted with(trimethylsilyl)acetylene to provide the (trimethylsilyl)ethynyl-substituted 3,4-dihydro-naphthalen-1(2H)-one compounds of Formula 8. Thereaction with (trimethylsilyl)acetylene is typically conducted underheat (approximately 100° C.) in the presence of cuprous iodide, asuitable catalyst, typically having the formula Pd(PPh₃)₂ Cl₂, an acidacceptor (such as triethylamine) under an inert gas (argon) atmosphere.Typical reaction time is approximately 24 hours. The(trimethylsilyl)ethynyl- substituted 3,4-dihydro-naphthalen-1 (2H)-onecompounds of Formula 8 are then reacted with base (potassium hydroxideor potassium carbonate) in an alcoholic solvent, such as methanol, toprovide the ethynyl substituted 3,4-dihydro-1-naphthalen-1(2H)ones ofFormula 9. Compounds of Formula 9 are then coupled with the aromatic orheteroaromatic reagent X₁ --Y(R₂)--A--B' (Formula 10) in the presence ofcuprous iodide, a suitable catalyst, typically Pd(PPh₃)₂ Cl₂, an acidacceptor, such as triethylamine, under inert gas (argon) atmosphere.Alternatively, a zinc salt (or other suitable metal salt) of thecompounds of Formula 9 can be coupled with the reagents of Formula 10 inthe presence of Pd(PPh₃)₄ or similar complex. Typically, the couplingreaction with the reagent X₁ --Y(R₂)--A--B' (Formula 10) is conducted atroom or moderately elevated temperature. Generally speaking, couplingbetween an ethynylaryl derivative or its zinc salt and a halogensubstituted aryl or heteroaryl compound, such as the reagent of Formula10, is described in U.S. Pat. No. 5,264,456, the specification of whichis expressly incorporated herein by reference. The compounds of Formula11 are precursors to exemplary compounds of the present invention, orderivatives thereof protected in the B' group, from which the protectinggroup can be readily removed by reactions well known in the art. Thecompounds of Formula 11 can also be converted into further precursors tothe exemplary compounds by such reactions and transformations which arewell known in the art. Such reactions are indicated in Reaction Scheme 1by conversion into "homologs and derivatives". One such conversionemployed for the synthesis of several exemplary compounds issaponification of an ester group (when B or B' is an ester) to providethe free carboxylic acid or its salt.

The halogen substituted aryl or heteroaryl compounds of Formula 10 can,generally speaking, be obtained by reactions well known in the art. Anexample of such compound is ethyl 4-iodobenzoate which is obtainable,for example, by esterification of 4-iodobenzoic acid. Another example isethyl 6-iodonicotinoate which can be obtained by conducting a halogenexchange reaction on 6-chloronicotinic acid, followed by esterification.Even more generally speaking, regarding derivatization of compounds ofFormula 11 and/or the synthesis of aryl and heteroaryl compounds ofFormula 10 which can thereafter be reacted with compounds of Formula 9,the following well known and published general principles and syntheticmethodology can be employed.

Carboxylic acids are typically esterified by refluxing the acid in asolution of the appropriate alcohol in the presence of an acid catalystsuch as hydrogen chloride or thionyl chloride. Alternatively, thecarboxylic acid can be condensed with the appropriate alcohol in thepresence of dicyclohexylcarbodiimide and dimethylaminopyridine. Theester is recovered and purified by conventional means. Acetals andketals are readily made by the method described in March, AdvancedOrganic Chemistry, 2nd Edition, McGraw-Hill Book Company, p. 810).Alcohols, aldehydes and ketones all may be protected by formingrespectively, ethers and esters, acetals or ketals by known methods suchas those described in McOmie, Plenum Publishing Press, 1973 andProtecting Groups, Ed. Greene, John Wiley & Sons, 1981.

To increase the value of n in the compounds of Formula 10 beforeaffecting the coupling reaction of Reaction Scheme 1 (where suchcompounds corresponding to Formula 10 are not available from acommercial source) aromatic or heteroaromatic carboxylic acids aresubjected to homologation by successive treatment under Arndt-Eistertconditions or other homologation procedures. Alternatively, derivativeswhich are not carboxylic acids may also be homologated by appropriateprocedures. The homologated acids can then be esterified by the generalprocedure outlined in the preceding paragraph.

Compounds of Formula 10, (or other intermediates or exemplary compounds)where A is an alkenyl group having one or more double bonds can be madefor example, by synthetic schemes well known to the practicing organicchemist; for example by Wittig and like reactions, or by introduction ofa double bond by elimination of halogen from an alpha-halo-arylalkyl-carboxylic acid, ester or like carboxaldehyde. Compounds of Formula 10(or other intermediates or exemplary compounds) where the A group has atriple (acetylenic) bond can be made by reaction of a correspondingaromatic methyl ketone with strong base, such as lithiumdiisopropylamide, reaction with diethyl chlorophosphate and subsequentaddition of lithium diisopropylamide.

The acids and salts derived from compounds of Formula 11 (or otherintermediates or exemplary compounds) are readily obtainable from thecorresponding esters. Basic saponification with an alkali metal basewill provide the acid. For example, an ester of Formula 11 (or otherintermediates or exemplary compounds) may be dissolved in a polarsolvent such as an alkanol, preferably under an inert atmosphere at roomtemperature, with about a three molar excess of base, for example,lithium hydroxide or potassium hydroxide. The solution is stirred for anextended period of time, between 15 and 20 hours, cooled, acidified andthe hydrolysate recovered by conventional means.

The amide may be formed by any appropriate amidation means known in theart from the corresponding esters or carboxylic acids. One way toprepare such compounds is to convert an acid to an acid chloride andthen treat that compound with ammonium hydroxide or an appropriateamine.

Alcohols are made by converting the corresponding acids to the acidchloride with thionyl chloride or other means (J. March, AdvancedOrganic Chemistry 2nd Edition, McGraw-Hill Book Company), then reducingthe acid chloride with sodium borohydride (March, Ibid, p. 1124), whichgives the corresponding alcohols. Alternatively, esters may be reducedwith lithium aluminum hydride at reduced temperatures. Alkylating thesealcohols with appropriate alky halides under Williamson reactionconditions (March, Ibid, p. 357) gives the corresponding ethers. Thesealcohols can be converted to esters by reacting them with appropriateacids in the presence of acid catalysts or dicyclohexylcarbodiimide anddimethylaminopyridine.

Aldehydes can be prepared from the corresponding primary alcohols usingmild oxidizing agents such as pyridinium dichromate in methylenechloride (Corey, E. J., Schmidt, G., Tet. Lett. 399, 1979), or dimethylsulfoxide/oxalyl chloride in methylene chloride (Omura, K., Swern, D.,Tetrahedron 34: 1651 (1978)).

Ketones can be prepared from an appropriate aldehyde by treating thealdehyde with an alkyl Grignard reagent or similar reagent followed byoxidation.

Acetals or ketals can be prepared from the corresponding aldehyde orketone by the method described in March, Ibid, p. 810.

Compounds of Formula 10 (or other intermediates, or exemplary compounds)where B is H can be prepared from the corresponding halogenated aromaticor hetero aromatic compounds, preferably where the halogen is I.

Referring back again to Reaction Scheme 1, the compounds of Formula 11are reacted with sodium bis(trimethylsilyl)amide and 2-N,N-bis(trifluoromethylsulfonyl)amino!-5-chloropyridine in an inertether type solvent, such as tetrahydrofuran, at low temperatures (-78°C. and 0°C.). This is shown in Reaction Scheme 1 where the usuallyunisolated sodium salt intermediate is shown in brackets as Formula 12.The reaction results in the trifluoromethylsulfonyloxy derivativesrepresented in Formula 13. (Tf═SO₂ CF₃). The compounds of Formula 13 arethen converted to the exemplary compounds of the invention, shown inFormula 14, by reaction with an organometal derivative derived from thearyl or heteroaryl compound R₁₄ H, such that the formula of theorganometal derivative is R₁₄ Met (Met stands for monovalent metal),preferably R₁₄ Li. (R₁₄ is defined as in connection with Formula 1.) Thereaction with the organometal derivative, preferably lithium derivativeof the formula R₁₄ Li is usually conducted in an inert ether typesolvent (such as tetrahydrofuran) in the presence of zinc chloride(ZnCl₂) and tetrakis(triphenylphosphine)-palladium(O) (Pd(PPh₃)₄). Theorganolithium reagent R₁₄ Li, if not commercially available, can beprepared from the compound R₁₄ H (or its halogen derivative R₁₄ -X₁where X₁ is halogen) in an ether type solvent in accordance with knownpractice in the art. The temperature range for the reaction between thereagent R₁₄ Li and the compounds of Formula 13 is, generally speaking inthe range of approximately -78° C. to 50° C. The compounds of Formula 14can be converted into further homologs and derivatives in accordancewith the reactions discussed above.

The intermediate 7-bromo-tetrahydronaphthalene-1- one compounds ofFormula 7 shown in Reaction Scheme 1 can also be converted with aGrignard reagent of the formula R₁₄ MgBr (R₁₄ is defined as inconnection with Formula 1) to yield the tertiary alcohol of Formula 15.The tertiary alcohol is dehydrated by treatment with acid to provide the3,4-dihydro-7-bromonaphthalene derivatives of Formula 16, which serve asintermediates for the synthesis of additional compounds of the presentinvention (see Reaction Schemes 6, 7, and 8). ##STR7##

Referring now to Reaction Scheme 2 a synthetic route to those compoundsis disclosed where with reference to Formula 1 X is S, O or NR' and theZ group is an ethynyl function (--C═--C--). Starting material for thissequence of the reaction is a bromophenol, bromothiophenol orbromoaniline of the structure shown in Formula 17. For the sake ofsimplifying the present specification, in the ensuing description X canbe considered primarily sulfur as for the preparation of benzothiopyranderivatives. It should be kept in mind, however, that the hereindescribed scheme is also suitable, with such modifications which will bereadily apparent to those skilled in the art, for the preparation ofbenzopyran (X═O) and dihydroquinoline (X═NR') compounds of the presentinvention. Thus, the compound of Formula 17, preferably parabromothiophenol, para bromophenol or para bromoaniline is reacted underbasic condition with a 3-bromo carboxylic acid of the Formula 18. Inthis reaction scheme the symbols have the meaning described inconnection with Formula 1. An example for the reagent of Formula 18where R₃ is hydrogen, is 3-bromopropionic acid. The reaction with the3-bromocarboxylic acid of Formula 18 results in the compound of Formula19. The latter is cyclized by treatment with acid to yield the6-bromothiochroman-4-one derivative (when X is S) or 6-bromochromanderivative (when X is 0) of Formula 20. The bromo compounds of Formula20 are then subjected to substantially the same sequence of reactionsunder analogous conditions, which are described in Reaction Scheme 1 forthe conversion of the bromo compounds of Formula 7 to the compounds ofthe invention. Thus, briefly summarized here, the bromo compounds ofFormula 20 are reacted with (trimethylsilyl)acetylene to provide the6-(trimethylsilyl)ethynyl-substituted thiochroman-4-one or chroman-4-onecompounds of Formula 21. The 6-(trimethylsilyl)ethynyl-substitutedthiochroman-4-one compounds of Formula 21 are then reacted with base(potassium hydroxide or potassium carbonate) to provide the ethynylsubstituted 6-ethynyl substituted thiochroman-4-ones of Formula 22.Compounds of Formula 22 are then coupled with the aromatic orheteroaromatic reagent X₁ -Y(R₂)-A-B' (Formula 10) under conditionsanalogous to those described for the analogous reactions of ReactionScheme 1, to yield the compounds of Formula 23.

The compounds of Formula 23 are then reacted still under conditionsanalogous to the similar reactions described in Reaction Scheme 1 withsodium bis(trimethylsilyl)amide and 2-N,N-bis(trifluoromethylsulfonyl)amino!-5-chloropyridine to yield the4-trifluoromethylsulfonyloxy benzothiopyran or benzopyran derivativesrepresented in Formula 24. The compounds of Formula 24 are thenconverted to compounds shown in Formula 25, by reaction with anorganometal derivative derived from the aryl or heteroaryl compound R₁₄H, as described in connection with Reaction Scheme 1.

Similarly to the use of the intermediate 7-bromo-tetrahydronaphthalene-1-one compounds of Formula 7 of Reaction Scheme 1,the intermediate 6-bromothiochroman-4-one compounds of Formula 20 canalso be used for the preparation of further compounds within the scopeof the present invention, as described below, in Reaction Schemes 6, 7and 8. The compounds of Formula 25, can also be converted into furtherhomologs and derivatives, in reactions analogous to those described inconnection with Reaction Scheme 1. ##STR8##

Reaction Scheme 3 discloses a synthetic route to compounds where, withreference to Formula 1, X is C(R₁)₂ !_(n), N is 0 and the Z group is anethynyl function (--C.tbd.C--). In accordance with this scheme, a6-bromo-2,3-dihydro-1H-inden-1-one derivative of Formula 26 is reactedin a sequence of reactions (starting with reaction withtrimethylsilylacetylene) which are analogous to the reactions describedabove in connection with Reaction Schemes 1 and 2, to provide, throughintermediates of the formulas 27-30, the indene derivatives of Formula31. In a preferred embodiment within the scope of Reaction Scheme 3, thestarting material is 6-bromo-2,3-dihydro-3,3-dimethyl-1H-inden-1-onethat is available in accordance with the chemical literature (See Smithet al. Org. Prep. Proced. Int. 1978 10, 123-131). Compounds of Formula26, such as 6-bromo-2,3-dihydro-3,3-dimethyl-1H-inden-1-one, can also beused for the synthesis of still further exemplary compounds for use inthe present invention, as described below. ##STR9##

Referring now to Reaction Scheme 4 a synthetic route to exemplarycompounds is disclosed where, with reference to Formula 1, Z is --(CR₁═CR₁)_(n),--, n' is 3, 4 or 5 and Y represents a direct valence bondbetween the (CR₁ ═CR₁)_(n), group and B. This synthetic route isdescribed for examples where the X group is C(R₁)₂ !_(n) and n is 1(dihydronaphthalene derivatives). Nevertheless, it should be understoodthat the reactions and synthetic methodology described in ReactionScheme 4 and further ensuing schemes, is also applicable, with suchmodifications which will be readily apparent to those skilled in theart, to derivatives where X is is S, O, NR' (benzothiopyran, benzopyranor dihydroquinoline derivatives) or C(R₁)₂ !_(n) and n is 0 (indenederivatives).

In accordance with Reaction Scheme 4, a 1,2,3,4-tetrahydronaphthalenederivative of Formula 32 is reacted with an acid chloride (R₁ COCl)under Friedel Crafts conditions, and the resulting acetylated product isoxidized, for example in a Jones oxidation reaction, to yield a mixtureof isomeric 6- and 7-acetyl-1(2H)-naphthalenone derivatives of Formula33. In a specific preferred example of this reaction, the startingcompound of Formula 32 is 1,2,3,4-tetrahydro-1,1-dimethylnaphthalene (aknown compound) which can be prepared in accordance with a processdescribed in the experimental section of the present application. The7-acetyl-1(2H)-naphthalenone derivative of Formula 33 is reacted withethylene glycol in the presence of acid to protect the oxo function ofthe exocyclic ketone moiety, as a ketal derivative of Formula 34. Theketal of Formula 34 is thereafter reacted with a Grignard reagent of theformula R₁₄ MgBr (the symbols are defined as in connection with Formula1), to yield the tertiary alcohol of Formula 35. Thereafter thedioxolane protective group is removed and the tertiary alcohol isdehydrated by treatment with acid to provide the 3,4-dihydro-7-acetylnaphthalene derivatives of Formula 36. The ketonefunction of the compounds of Formula 36 is subjeceted to a Horner Emmons(or analogous) reaction under strongly alkaline conditions with aphosphonate reagent of Formula 37, to yield, after reduction, thealdehyde compounds of Formula 38. Still another Homer Emmons (oranalogous) reaction under strongly alkaline conditions with a reagent ofFormula 39 provides compounds of Formula 40. The latter can be convertedinto further homologs and derivatives in accordance with the reactionsdescribed above. A specific example of the Homer Emmons reagent ofFormula 37 which is used for the preparation of a preferred compound isdiethylcyanomethylphosphonate; an example of the Horner Emmons reagentof Formula 39 is diethyl-(E)-3-ethoxycarbonyl-2-methylallylphosphonate.##STR10##

Reaction Scheme 5 discloses a synthetic process for preparing compoundswhere the Z group is an azo group (--N═N--). As in Reaction Scheme 4this process is described for examples where the X group is C(R₁)₂ !_(n)and n is 1 (dihydronaphthalene derivatives). Nevertheless, it should beunderstood that the synthetic methodology described is also applicable,with such modifications which will be readily apparent to those skilledin the art, to all azo compounds for use in the invention, namely toderivatives where X is is S, O, NR' (benzothiopyran, benzopyran ordihydroquinoline derivatives) or C(R₁)₂ !_(n) and n is 0 (indenederivatives). Thus, a nitro group is introduced into the startingcompound of Formula 6 under substantially standard conditions ofnitration, to yield the 3,4-dihydro-7-nitro-1(2H)-naphthalenonederivative of Formula 41. The latter compound is reduced to the3,4-dihydro-7-amino-1(2H)-naphthalenone derivative of Formula 42 and isthereafter reacted with a nitroso compound of the formula ON-Y(R₂)-A-B(Formula 43) under conditions normally employed (glacial acetic acid)for preparing azo compounds. The nitroso compound of Formula 43 can beobtained in accordance with reactions known in the art. A specificexample for such compound, which is used for the synthesis of apreferred compound is ethyl 4-nitrosobenzoate. The azo compound ofFormula 44 is thereafter reacted with sodium bis(trimethylsilyl)amideand 2- N,N-bis(trifluoromethylsulfonyl)amino!-5-chloropyridine to yieldthe 4-trifluoromethylsulfonyloxy derivatives represented in Formula 45.The compounds of Formula 45 are then converted to the azo compoundsshown in Formula 46, by reaction with an organometalic derivativederived from the aryl or heteroaryl compound R₁₄ H. These latter tworeactions, namely the conversion to the 4-trifluoromethylsulfonyloxyderivatives and subsequent reaction with the organometal derivative,have been described above in connection with Reaction Schemes 1, 2 and3, and are employed in several presently preferred synthetic processesleading to exemplary RAR antagonist compounds. ##STR11##

Reaction Scheme 6 discloses a presently preferred synthetic process forthe preparation of compounds where, with reference to Formula 1, the Zgroup is COO-- or CONR₁ (R₁ is preferably H). These ester and amidederivatives are prepared from the 3,4-dihydro-7- bromonaphthalenederivatives of Formula 16, which can be obtained as described inReaction Scheme 1. Thus, the compounds of Formula 16 are reacted withstrong base, such as t-butyllithium, in an inert ether type solvent,such as tetrahydrofuran, at cold temperature, and carbon dioxide (CO₂)is added to provide the 5,6- dihydro-2-naphthalenecarboxylic acidderivatives of Formula 47. Compounds of Formula 47 are then reacted withcompounds of the formula X₂ --Y(R₂)--A--B (Formula 48) where X₂reperesent an OH or an NR, group, the R₁ preferably being hydrogen.Those skilled in the art will recognize that the compounds of Formula 48are aryl or heteroaryl hydroxy or amino derivatives which can beobtained in accordance with the state-of-the-art. The reaction betweenthe compounds of Formula 47 and Formula 48 can be conducted undervarious known ester or amide forming conditions, such as coupling of thetwo in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride and 4-dimethylaminopyridine. Alternatively, the compoundsof Formula 47 can be converted into the corresponding acid chloride forcoupling with the compounds of Formula 48 in the presence of base. Theamide or ester compounds of Formula 49 can be converted into furtherhomologs and derivatives, as described above. Although Reaction Scheme 6is described and shown for the example where the X group of Formula 1 isC(R₁)₂ !n and n is 1 (dihydronaphthalene derivatives), the hereindescribed process can be adapted for the preparation of benzopyran,benzothiopyran, dihydroquinoline and indene derivatives as well.

Compounds of the present invention where with reference to Formula 1, Zis --OCO--, NR₁ CO, as well as the corresponding thioester and thioamideanalogs, can be prepared from the intermediates derived from thecompounds of Formula 16 where the bromo function is replaced with anamino or hydroxyl group and in accordance with the teachings of U.S.Pat. Nos. 5,324,744, the specification of which is expresslyincorporated herein by reference. ##STR12##

Reaction Scheme 7 discloses a presently preferred synthetic process forthe preparation of compounds where with reference to Formula 1, Z is--(CR₁ ═CR₁)_(n) -- and n' is 0. These compounds of Formula 50 can beobtained in a coupling reaction between compounds of Formula 16 and aGrignard reagent derived from the halo compounds of Formula 10. Thecoupling reaction is typically conducted in the presence of a zinc saltand a nickel (Ni(0)) catalyst in inert ether type solvent, such astetrahydrofuran. The compounds of Formula 50 can be converted intofurther homologs and derivatives, as described above. ##STR13##

Referring now to Reaction Scheme 8 a presently preferred syntheticprocess is disclosed for the preparation of compounds where Z is --(CR₁═CR₁)_(n),-- and n' is 1. More particularly, Reaction Scheme 8 disclosesthe presently preferred process for preparing those compounds which aredihydronaphtalene derivatives and where the Z group represents a vinyl(--CH═CH--) function. However, the generic methodology disclosed hereincan be extended, with such modifications which will be apparent to thoseskilled in the art, to the analogous benzopyran, benzothiopyran,dihydroquinoline compounds, and to compounds where the vinyl group issubstituted. Thus, in accordance with Reaction Scheme 8 the7-bromo-1(2H)-naphthalenone derivative of Formula 7 is reacted with avinyl derivative of the structure --CH₂ ═CH--Y(R₂)--A--B (Formula 51) inthe presence of a suitable catalyst, typically having the formulaPd(PPh₃), an acid acceptor (such as triethylamine) under an inert gas(argon) atmosphere. The conditions of this reaction are analogous to thecoupling of the acetylene derivatives of Formula 9 with the reagent ofFormula 10 (see for example Reaction Scheme 1), and this type ofreaction is generally known in the art as a Heck reaction. The vinylderivative of Formula 51 can be obtained in accordance with the state ofthe art, an example for such a reagent used for the synthesis of apreferred compound to be used in the invention is ethyl 4-vinylbenzoate.

The product of the Heck coupling reaction is an ethenyl derivative ofFormula 52, which is thereafter converted into compounds used in thepresent invention by treatment with sodium bis(trimethylsilyl)amide and2- N,N-bis(trifluoromethylsulfonyl)amino!-5-chloropyridine to yield the4-trifluoromethylsulfonyloxy derivatives of Formula 53, and subsequentreaction with an organometal derivative derived from the aryl orheteroaryl compound R₁₄ H, as described above. The resulting compoundsof Formula 54 can be converted into further homologs and derivatives.

The compounds of Formula 54 can also be obtained through syntheticschemes which employ a Wittig or Horner Emmons reaction. For example,the intermediate of Formula 33 (see Reaction Scheme 4) can be reactedwith a triphenylphosphonium bromide (Wittig) reagent or more preferablywith a diethylphosphonate (Homer Emmons) reagent of the structure (EtO)₂PO--CH₂ ----Y(R₂)--A--B, as described for analogous Homer Emmonsreactions in U.S. Pat. No. 5,324,840, the specification of which isincorporated herein by reference. The just mentioned Homer Emmonsreaction provides intermediate compounds analogous in structure toFormula 52, and can be converted into compounds of Formula 54 by thesequence of reactions described in Reaction Scheme 8 for the compoundsof Formula 52.

Synthetic Methods--Aryl and (3-Oxy-1-Propenyl)-Substituted Compounds

The exemplary RAR antagonist compounds of Formula 101 can be made by thesynthetic chemical pathways illustrated here. The synthetic chemist willreadily appreciate that the conditions set out here are specificembodiments which can be generalized to any and all of the compoundsrepresented by Formula 101. ##STR14##

Reaction Scheme 101 illustrates the synthesis of compounds of Formula101 where X is C(R₁)₂ !_(n), n is 1, p is zero and R₁₇ is H or loweralkyl. In other words, Reaction Scheme 101 illustrates the synthesis ofcompounds of the invention which are 3,4-dihydronaphthalene derivatives.In accordance with this scheme, a tetrahydronaphthalene compound ofFormula 103 which is appropriately substituted with the R₃ and R₂ groups(as these are defined in connection with Formula 101) serves as thestarting material. A preferred example of a compound of Formula 103 is1,3,3,4-tetrahydro-1,1-dimethyl-naphthalene, which is described in thechemical literature (Mathur et al. Tetrahedron, 1985, 41: 1509. Apresently preferred route for the synthesis of this compound from1-bromo-3-phenylpropane is also described in the experimental section ofthe present application.

The compound of Formula 103 is reacted in a Friedel Crafts type reactionwith an acid chloride having the structure R₁₆ CH₂ COCl (R₁₆ is definedas in connection with Formula 101) and is thereafter oxidized withchromium trioxide in acetic acid to provide the isomeric 6 and 7acyl-3,4-dihydro-1(2H)-naphthalenone derivatives. Only the 6-acylderivative which is of interest from the standpoint of the presentinvention, is shown by structural formula (Formula 104) in ReactionScheme 101. In the preparation of the presently preferred compounds ofthis invention the R₁ groups represent methyl, R₂, R₃ and R₁₆ are H, andtherefore the preferred intermediate corresponding to Formula 104 is3,4-dihydro-4,4-dimethyl-6-acetyl-1(2H)-naphthalenone.

The exocyclic ketone function of the compound of Formula 104 isthereafter protected as a ketal, for example by treatment with ethyleneglycol in acid, to provide the 1,3-dioxolanyl derivative of Formula 105.The compound of Formula 105 is then reacted with a Grignard reagent ofthe formula R₁₄ MgBr (R₁₄ is defined as in connection with Formula 101)to give the 1,2,3,4-tetrahydro-1-hydroxy-naphthalene derivative ofFormula 106. The exocyclic ketone function of the compound of Formula106 is then deprotected by treatment with acid and dehydrated to givethe compound of Formula 107.

An alternate method for obtaining the compounds of Formula 107 from thecompounds of Formula 105 is by reacting the compounds of Formula 105with sodium bis(trimethylsilyl)amide and 2-N,N-bis(trifluoromethylsulfonyl)amino!-5-chloropyridine (Tf═SO₂ CF₃) inan inert ether type solvent, such as tetrahydrofuran, at lowtemperatures (-78° C. and 0° C.). This reaction proceeds through asodium salt intermediate which is usually not isolated and is not shownin Reaction Scheme 101. The overall reaction results in atrifluoromethylsulfonyloxy derivative, which is thereafter reacted withan organometal derivative derived from the aryl or heteroaryl compoundR₁₄ H, such that the formula of the organometal derivative is R₁₄ Met(Met stands for monovalent metal), preferably R₁₄ Li. (R₁₄ is defined asin connection with Formula 101.) The reaction with the organometalderivative, preferably lithium derivative of the formula R₁₄ Li isusually conducted in an inert ether type solvent (such astetrahydrofuran) in the presence of zinc chloride (ZnCl₂) andtetrakis(triphenylphosphine)-palladium(0) (Pd(PPh₃)₄). The organolithiumreagent R₁₄ Li, if not commercially available, can be prepared from thecompound R₁₄ H (or its halogen derivative R₁₄ --X₁ where X₁ is halogen)in an ether type solvent in accordance with known practice in the art.The temperature range for the reaction between the reagent R₁₄ Li andthe trifluoromethylsulfonyloxy derivative is, generally speaking, in therange of approximately -78° C. to 50° C.

The compounds of the invention are formed as a result of a condensationbetween the ketone compound of Formula 107 and an aldehyde or ketone ofFormula 108. In the preparation of the preferred exemplary compounds ofthe invention the reagent of Formula 108 is 4-carboxybenzaldehyde (R₁₇--H). Examples of other reagents within the scope of Formula 108 andsuitable for the condensation reaction and for the synthesis ofcompounds within the scope of the present invention (Formula 101) are:5-carboxy-pyridine-2-aldehyde, 4-carboxy-pyridine-2-aldehyde,4-carboxy-thiophene-2-aldehyde, 5-carboxy-thiophene-2-aldehyde,4-carboxy-furan-2-aldehyde, 5-carboxy-furan-2-aldehyde,4-carboxyacetophenone, 2-acetyl-pyridine-5-carboxylic acid,2-acetyl-pyridine-4-carboxylic acid, 2-acetyl- thiophene-4-carboxylicacid, 2-acetyl-thiophene-5-carboxylic acid, 2-acetyl-furan-4-carboxylicacid, and 2-acetyl-furan-5-carboxylic acid. The latter compounds areavailable in accordance with the chemical literature; see for exampleDecroix et al., J Chem. Res.(S), 4: 134 (1978); Dawson et al., J Med.Chem. 29: 1282 (1983); and Queguiner et al., Bull Soc. Chimique deFrance No. 10, pp. 3678-3683 (1969). The condensation reaction betweenthe compounds of Formula 107 and Formula 108 is conducted in thepresence of base in an alcoholic solvent. Preferably, the reaction isconducted in ethanol in the presence of sodium hydroxide. Those skilledin the art will recognize this condensation reaction as an aldolcondensation, and in case of the herein described preferred examples(condensing a ketone of Formula 107 with an aldehyde of Formula 108) asa Claisen-Schmidt reaction. (See March: Advanced Organic Chemistry:Reactions Mechanisms, and Structure, pp. 694-695 McGraw Hill (1968). Thecompounds of Formula 109 are within the scope of the present invention,and can also be subjected to further transformations resulting inadditional compounds of the invention. Alternatively, the A-B group ofFormula 108 may be a group which is within the scope of the invention,as defined in Formula 101, only after one or more synthetictransformations of such a nature which is well known and within theskill of the practicing organic chemist. For example, the reactionperformed on the A-B group may be a deprotection step, homologation,esterification, saponification, amide formation or the like.

Generally speaking, regarding derivatization of compounds of Formula 109and/or the synthesis of aryl and heteroaryl compounds of Formula 108which can thereafter be reacted with compounds of Formula 107, thefollowing well known and published general principles and syntheticmethodology can be employed.

As indicated above, carboxylic acids are typically esterified byrefluxing the acid in a solution of the appropriate alcohol in thepresence of an acid catalyst such as hydrogen chloride or thionylchloride. Alternatively, the carboxylic acid can be condensed with theappropriate alcohol in the presence of dicyclohexylcarbodiimide anddimethylaminopyridine. The ester is recovered and purified byconventional means. Acetals and ketals are readily made by the methoddescribed in March, Advanced Organic Chemistry 2nd Edition, McGraw-HillBook Company, p. 810). Alcohols, aldehydes and ketones all may beprotected by forming respectively, ethers and esters, acetals or ketalsby known methods such as those described in McOmie, Plenum PublishingPress, 1973 and Protecting Groups, Ed. Greene, John Wiley & Sons, 1981.

To increase the value of n in the compounds of Formula 108 beforeaffecting the condensation reaction of Reaction Scheme 101 (where suchcompounds corresponding to Formula 108 are not available from acommercial source) aromatic or heteroaromatic carboxylic acids may besubjected to homologation (while the aldehyde group is protected) bysuccessive treatment under Arndt- Eistert conditions or otherhomologation procedures. Alternatively, derivatives which are notcarboxylic acids may also be homologated by appropriate procedures. Thehomologated acids can then be esterified by the general procedureoutlined in the preceding paragraph.

Compounds of Formula 108, (or other intermediates or of the invention,as applicable) where A is an alkenyl group having one or more doublebonds can be made for example, by synthetic schemes well known to thepracticing organic chemist; for example by Wittig and like reactions, orby introduction of a double bond by elimination of halogen from analpha-halo-arylalkyl- carboxylic acid, ester or like carboxaldehyde.Compounds of Formula 108 (or other intermediates or of the invention, asapplicable) where the A group has a triple (acetylenic) bond can be madeby reaction of a corresponding aromatic methyl ketone with strong base,such as lithium diisopropylamide, reaction with diethyl chlorophosphateand subsequent addition of lithium diisopropylamide.

The acids and salts derived from compounds of Formula 109 (or otherintermediates or compounds of the invention, as applicable) are readilyobtainable directly as a result of the condensation reaction, or fromthe corresponding esters. Basic saponification with an alkali metal basewill provide the acid. For example, an ester of Formula 109 (or otherintermediates or compounds of the invention, as applicable) may bedissolved in a polar solvent such as an alkanol, preferably under aninert atmosphere at room temperature, with about a three molar excess ofbase, for example, lithium hydroxide or potassium hydroxide. Thesolution is stirred for an extended period of time, between 15 and 20hours, cooled, acidified and the hydrolysate recovered by conventionalmeans.

The amide may be formed by any appropriate amidation means known in theart from the corresponding esters or carboxylic acids. One way toprepare such compounds is to convert an acid to an acid chloride andthen treat that compound with ammonium hydroxide or an appropriateamine.

Alcohols are made by converting the corresponding acids to the acidchloride with thionyl chloride or other means (J. March, AdvancedOrganic Chemistry 2nd Edition, McGraw-Hill Book Company), then reducingthe acid chloride with sodium borohydride (March, Ibid, p. 1124), whichgives the corresponding alcohols. Alternatively, esters may be reducedwith lithium aluminum hydride at reduced temperatures. Alkylating thesealcohols with appropriate alky halides under Williamson reactionconditions (March, Ibid, p. 357) gives the corresponding ethers. Thesealcohols can be converted to esters by reacting them with appropriateacids in the presence of acid catalysts or dicyclohexylcarbodiimide anddimethylaminopyridine.

Aldehydes can be prepared from the corresponding primary alcohols usingmild oxidizing agents such as pyridinium dichromate in methylenechloride (Corey, E. J., Schmidt, G., Tet. Lett., 399, 1979), or dimethylsulfoxide/oxalyl chloride in methylene chloride (Omura, K., Swern, D.,Tetrahedron, 34: 1651 (1978)).

Ketones can be prepared from an appropriate aldehyde by treating thealdehyde with an alkyl Grignard reagent or similar reagent followed byoxidation.

Acetals or ketals can be prepared from the corresponding aldehyde orketone by the method described in March, Ibid, p. 810. ##STR15##

Referring now to Reaction Scheme 102, a synthetic route to thosecompounds of the invention is described in which, with reference toFormula 101 p is zero, R₂ in the aromatic portion of the condensed ringstructure is OH and R₁₇ is OH. Those skilled in the art will readilyrecognize that these compounds are β-diketones in the enol form.Reaction Scheme 102 also describes a synthetic route to those compoundsof the invention where p is 1. Those skilled in the art will readilyrecognize that the latter compounds are flavones. Thus, in accordancewith this scheme a 1,2,3,4-tetrahydro-6-methoxynaphthalene-1-onederivative of Formula 110 is reacted with dialkyl zinc (R₁ Zn) in thepresence of titanium tetrachloride in a suitable solvent such as CH₂ Cl₂to replace the oxo function with the geminal dialkyl group R₁ R₁, toyield a compound of Formula 111, where R₁ is lower alkyl. In preferredembodiments of the compounds of the invention which are made inaccordance with Reaction Scheme 102 the R₃ group is hydrogen and R₁ aremethyl. Accordingly, the dialkyl zinc reagent is dimethyl zinc, and thepreferred starting material of Formula 110 is1,2,3,4-tetrahydro-6-methoxynaphthalene-1 -one. The latter compound iscommercially available, for example from Aldrich Chemical Company. The1,2,3,4-tetrahydro-1,2-dialkyl-6-methoxy naphthalene derivative ofFormula 111 is thereafter oxidized with chromium trioxide in acetic acidand acetic anhydride to give a 1,2,3,4-tetrahydro-3,4-dialkyl-7-methoxynaphthalen-1-one derivative of Formula 112. The ketone compound ofFormula 112 is reacted with a Grignard reagent (R₁₄ MgBr, R₁₄ is definedas in connection with Formula 101) to yield a1-hydroxy-1-aryl-3,4-dihydro-3,4-dialkyl-7-methoxy naphthalenederivative of Formula 113. The hydroxy compound of Formula 113 issubjected to elimination by heating, preferably in acid, to yield thedihydronaphthalene compound of Formula 114. The methyl group is removedfrom the phenolic methyl ether function of the compound of Formula 114by treatment with boron tribromide in a suitable solvent, such as CH₂Cl₂, and therafter the phenolic OH is acylated with an acylating agentthat introduces the R₁₆ CH₂ CO group, to give a compound of Formula 115.In the preferred embodiment R₁₆ is H, and therefore the acylating agentis acetyl chloride or acetic anhydride. The acetylation reaction isconducted in a basic solvent, such as pyridine. The acylated phenolcompound of Formula 115 is reacted with aluminum chloride at elevatedtemperature, causing it to undergo a Fries rearrangement and yield the1-aryl-3,4-dialkyl-3,4-dihydro-6-acyl-7-hydroxy-naphthalene compound ofFormula 116. The phenolic hydroxyl group of the compound of Formula 116is acylated with an acylating agent (such as an acid chloride) thatintroduces the CO-Y(R₂)-A-B group to yield a compound of Formula 117. Inthe acid chloride reagent Cl-CO-Y(R₂)-A-B (or like acylating reagent)the symbols Y, R₂ and A-B have the meaning defined in connection withFormula 101. In the preparation of a preferred compound of the inventionin accordance with this scheme this reagent is ClCOC₆ H₄ COOEt (the halfethyl ester half acid chloride of terephthalic acid).

The compound of Formula 117 is reacted with strong base, such aspotassium hydroxyde in pyridine, to yield, as a result of anintramolecular Claisen condensation reaction, a compound of Formula 118.The compounds of Formula 118 are within the scope of the invention andof Formula 101, where there is an OH for the R₂ substituent in thearomatic portion of the condensed ring moiety and R₁₇ is OH. Inconnection with the foregoing reaction (intramolecular Claisencondensation) and the previously mentioned Fries rearrangement it isnoted that these probable reaction mechanisms are mentioned in thisdescription for the purpose of fully explaining the herein describedreactions, and for facilitating the work of a person of ordinary skillin the art to perform the herein described reactions and prepare thecompounds of the invention. Nevertheless, the present inventors do notwish to be bound by reaction mechanisms and theories, and the hereinclaimed invention should not be limited thereby.

The compounds of Formula 118 are reacted with strong acid, such assulfuric acid, in a suitable protonic solvent, such as acetic acid, toyield the flavone compounds of Formula 119. The compounds of Formula 119are also compounds of the invention, within the scope of Formula 101where p is 1. Both the compounds of Formula 118 and Formula 119 can besubjected to further reactions and transformations to provide furtherhomologs and derivatives, as described above in connection with ReactionScheme 101. This is indicated in Reaction Scheme 102 as conversion tohomologs and derivatives. ##STR16##

Referring now to Reaction Scheme 103 a synthetic route is shown leadingto those compounds of the invention where, with reference to Formula 101X is S, O or NR', p is zero and R₁₇ is H or lower alkyl. However, byapplying the generic principles of synthesis shown in Reaction Scheme102 the presently shown synthetic process can be modified or adapted bythose of ordinary skill in the art to also obtain compounds of theinvention where X is S, O or NR' and p is 1, or where X is S, O or NR'and p is zero, the R₂ group in the aromatic portion of the condensedring moiety is OH and R₁₇ is OH.

The starting compound of Reaction Scheme 103 is a phenol, thiophenol oraniline derivative of Formula 120. In the presently preferred compoundsof the invention the R₂ and R₁₆ groups are both hydrogen, and thepreferred starting compounds of Formula 120 are 3-ethenyl-thiophenol or3-ethenyl-phenol which are known in the chemical literature (Nuyken, etal. Polym. Bull (Berlin) 11: 165 (1984). For the sake of simplifying thepresent specification, in the ensuing description X can be consideredprimarily sulfur as for the preparation of benzothiopyran derivatives ofthe present invention. It should be kept in mind, however, that theherein described scheme is also suitable, with such modifications whichwill be readily apparent to those skilled in the art, for thepreparation of benzopyran (X═O) and dihydroquinoline (X═NR') compoundswithin the scope of the present invention. Thus, the compound of Formula120 is reacted under basic condition with a 3-bromo carboxylic acid ofthe Formula 121. In this reaction scheme the symbols have the meaningdescribed in connection with Formula 101. An example for the reagent ofFormula 121 where R₃ is hydrogen, is 3-bromopropionic acid. The reactionwith the 3-bromocarboxylic acid of Formula 121 results in the compoundof Formula 122. The latter is cyclized by treatment with acid to yieldthe 7-ethenyl-thiochroman-4-one derivative (when X is S) or7-ethenyl-chroman derivative (when X is 0) of Formula 123. The7-ethenyl- thiochroman-4-one or 7-ethenyl-chroman-4-one derivative ofFormula 123 is oxidized in the presence of Pd(II)C₂ and CuC₂ catalyststo provide the corresponding 7-acyl (ketone) compound of Formula 124.Those skilled in the art will recognize the latter reaction as a Wackeroxidation. The exocyclic ketone group of the compound of Formula 124 isprotected in the form of a ketal, for example by treatment with ethyleneglycol in acid, to provide the 1,3-dioxolanyl derivative of Formula 125.Thereafter the compound of Formula 125 is subjected to a sequence ofreactions analogous to those described for the compounds of Formula 105in Reaction Scheme 101. Thus, the 1,3- dioxolanyl derivative of Formula125 is reacted with a Grignard reagent of the formula R₁₄ MgBr to givethe tertiary alcohol of Formula 126, which is thereafter dehydrated inacid to provide the benzothiopyran (X is S), benzopyran (X is O) ordihydroquinoline (X is NR') derivative of Formula 127. The ketonecompound of Formula 127 is then reacted in the presence of base with thereagent of Formula 108 in an aldol condensation (Claisen-Schmidt)reaction to provide compounds of the invention of Formula 128. Thecompounds of Formula 128 can be converted into further homologs andderivatives, as described above in connection with Reaction Schemes 101and 102.

Specific Examples

2-hydroxy-2-methyl-5 -phenalpentane

To a mixture of magnesium turnings 13.16 g (0.541 mol) in 200 ml ofanhydrous Et₂ O was added 100.0 g (0.492 mol) of 1-bromo-3-phenylpropane as a solution in 100 ml of Et20. After of 5-10 ml of thesolution had been added, the addition was stopped until the formation ofthe Grignard reagent was in progress. The remaining bromide was thenadded over 1 hour. The Grignard reagent was stirred for 20 minutes at35° C. and then 31.64 g (0.541 mol) of acetone was added over a 45minute period. The reaction was stirred overnight at room temperaturebefore being cooled to 0° C. and acidified by the careful addition of20% HCl. The aqueous layer was extracted with Et₂ O (3×200 ml) and thecombined organic layers washed with water, and saturated aqueous NaClbefore being dried over MgSO₄. Removal of the solvent under reducedpressure and distillation of the residue afforded 63.0 g (72%) of theproduct as a pale-yellow oil, bp 99°-102° C./0.5 mm Hg. 1H NMR (CDCl₃):δ7.28-7.18 (5H, m), 2.63 (2H, t, J=7.5 Hz), 1.68 (2H, m), 1.52 (2H, m),1.20 (6H, s).

1,2,3,4-tetrahydro-1,1-dimethylnaphthalene

A mixture of P₂ O₅ (55.3 g, 0.390 mol) in 400 ml of methanesulfonic acidwas heated to 105° C. under argon until all of the solid had dissolved.The resulting solution was cooled to room temperature and2-hydroxy-2-methyl-5-phenylpentane (63.0 g, 0.354 mol) added slowly withstirring. After 4 hours the reaction was quenched by carefully pouringthe solution onto 1 L of ice. The resulting mixture was extracted withEt₂ O (4×125 ml) and the combined organic layers washed with water,saturated aqueous NaHCO₃, water, and saturated aqueous NaCl before beingdried over MgSO₄. Concentration of the solution under reduced pressure,followed by distillation afforded 51.0 g (90%) of the product as a clearcolorless oil, bp. 65°-67° C./1.1 mmHg. 1H NMR (CDCl₃): δ7.32 (1H, d,J=7.4 Hz), 7.16-7.05 (3H, m), 2.77 (2H, t, J =6.3 Hz), 1.80 (2H, m),1.66 (2H, m), 1.28 (6H, s).

3,4-dihydro-4,4-dimethyl-1 (2H)-naphthalenone (Compound A)

A solution of 350 ml of glacial acetic acid and 170 ml of aceticanhydride was cooled to 0° C. and CrO₃, 25.0 g (0.25 mol) carefullyadded in small portions. The resulting mixture was stirred for 30minutes before 120 ml of benzene was added.1,2,3,4-tetrahydro-1,1-dimethylnaphthalene was added slowly as asolution in 30 ml of benzene. Upon completing the addition the reactionwas stirred for 4 hours at 0° C. The solution was diluted with H₂ O (200ml) and extracted with Et₂ O (5×50 ml). The combined organic layers werewashed with water, saturated aqueous NaCO₃, and saturated aqueous NaCl,before being dried over MgSO₄. Removal of the solvents under reducedpressure, and distillation afforded 16.0 g (74%) of the product as apale-yellow oil, bp 93°-96° C./0.3 mm Hg 1H NMR (CDCl₃): δ8.02 (1H, dd,J 1.3, 7.8 Hz), 7.53 (1H, m ), 7.42 (1H, d, J=7.9 Hz), 7.29 (1H, m),2.74 (2H, t, J=6.8 Hz), 2.02 (2H, t, J=6.8 Hz), 1.40 (6H, s).

3,4-dihydro-4,4-dimethyl-7-bromo-1 (2H)-naphthalenone (Compound B)

A 100 ml three-necked flask, fitted with an efficient reflux condenserand drying tube, and addition funnel, was charged with a mixture ofAlCl₃ 9.5g (71.4 mmol) and 3 ml of CH₂ Cl₂. The3,4-dihydro-4,4-dimethyl-1(2H)-naphthalenone (5.0 g, 28.7 mmol), wasadded dropwise with stirring (Caution: Exothermic Reaction|) to themixture at room temperature. Bromine, 5.5 g (34.5 mmol), was then addedvery slowly, and the resulting mixture stirred for 2 hours at roomtemperature. (Note: if stirring stops, the mixture can be warmed to 70°C. until stirring resumes.) The reaction was then quenched by the slowaddition of ice-cold 6M HCl. The mixture was extracted with Et₂ O andthe combined organic layers washed with water, saturated aqueous NaHCO₃,and saturated NaCl, before being dried over MgSO₄. Removal of thesolvent under reduced pressure, and distillation of the residue afforded5.8 g (80%) of the product as a pale-yellow oil which solidified onstanding, bp: 140° C./0.4 mm Hg. 1H NMR (CDCl₃): δ8.11 (1H, d, J=3.0Hz), 7.61 (1H, dd, J=3.0, 9.0 Hz), 7.31 (1H, d, J=9.0 Hz), 2.72 (2H, t,J=6.0 Hz), 2.01 (2H, t, J=6.0 Hz), 1.28 (6H, s).

1,2,3,4-tetrahydro-1 -hydroxy-1-(4-methylphenyl)-4,4-dimethyl-7-bromonaphthalene (Compound C)

To a mixture of magnesium turnings (648.0 mg, 27.0 mmol) in 25 ml of THFwas added a solution of 4-bromotoluene (5.40 g, 31.8 mmol) in 10 ml ofTHF in two portions. The reaction was initiated by the addition of 2 mlof the solution, followed by the slow addition of the remaining solutionvia an addition funnel. The mixture was stirred at room temperature for1 hour, and then the solution was transferred to a second flask using acanula. To the resulting Grignard reagent was added 4.0 g (15.9 mmol) of3,4-dihydro-4,4-dimethyl-7-bromo-1(2H)-naphthalenone (Compound B) as asolution in 15 ml of THF. The resulting solution was heated to refluxovernight, cooled to room temperature, and the reaction quenched by thecareful addition of ice-cold 10% HCl. Extraction with Et₂ O was followedby washing of the combined organic layers with H₂ O and saturatedaqueous NaCl, then drying over MgSO₄. Removal of the solvent underreduced pressure provided an oil which afforded the product as acolorless solid after column chromatography (hexanes/EtOAc, 96:4). 1HNMR (CDCl₃): δ7.36 (1H, dd, J=2.1, 7.6 Hz), 7.26 (3H, m), 7.12 (3H, s),2.34 (3H, s), 2.24-2.04 (2H, m), 1.81 (1H, m), 1.55 (1H, m), 1.35 (3H,s), 1.30 (3H, s).

3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-7- bromonaphthalene(Compound D)

A flask equipped with a Dean-Stark trap was charged with 3.4 g of (9.85mmol) of 1,2,3,4-tetrahydro-1 -hydroxy-1-(4-methylphenyl)-4,4-dimethyl-7-bromonaphthalene (Compound C) and 40 mlof benzene. A catalytic amount of p-toluenesulfonic acid monohydrate wasadded and the resulting solution heated to reflux for 2 hours. Uponcooling to room temperature, Et₂ O was added and the solution washedwith H₂ O, saturated aqueous NaHCO₃, and saturated aqueous NaCl thendried over MgSO₄. Removal of the solvents under reduced pressure, andcolumn chromatography (100% hexane/silica gel) afforded the titlecompound as a colorless solid. 1H NMR (CDCl₃): δ7.32 (1H, dd, J=2.1, 8.2Hz), 7.21 (5H, m), 7.15 (1H, d, J=2.1 Hz), 5.98 (1H, t, J=4.7 Hz), 2.40(3H, s), 2.32 (2H, d, J=4.7 Hz), 1.30 (6H, s).

7-Ethynyl-3 4-dihydro-4,4-dimethylnaphthalene-1 (2H)-one (Compound E)

To a solution (flushed for 15 minutes with a stream of argon) of 7 g(27.6 mmol) of 3,4-dihydro-4,4-dimethyl-7-bromo-1(2H)-naphthalenone(Compound B) in 150 ml of triethylamine was added 0.97 g (1.3 mmol) ofbis(triphenylphosphine)palladium(II) chloride and 0.26 g (1.3 mmol) ofcuprous iodide. The solution mixture was flushed with argon for 5minutes and then 39 ml (36.6 mmol) of (trimethylsilyl)acetylene wasadded. The reaction mixture was sealed in a pressure tube and placed ina preheated oil bath (100° C.) for 24 hours. The reaction mixture wasthen filtered through Celite, washed with Et₂ O and the filtrateconcentrated in vacuo to give crude7-(trimethylsilyl)ethynyl-3,4-dihydro-4,4-dimethylnaphthalen-1(2H)-one.To a solution of this crude TMS-acetylenic compound in 50 ml of methanolwas added 0.6 g (4.3 mmol) of K₂ CO₃. The mixture was stirred for 8hours at ambient temperature and then filtered. The filtrate wasconcentrated in vacuo, diluted with Et₂ O, washed with water, 10% HCland brine, dried over MgSO₄ and concentrated in vacuo. Purification bycolumn chromatography (silica, 10% EtOAc-hexane) yielded the titlecompound as a white solid. PMR (CDCl₃) : δ1.39 (6H, s), 2.02 (2H, t,J=7.0 Hz), 2.73 (2H, t, J=7.0 Hz), 3.08 (1H, s), 7.39 (1H, d, J=8.2 Hz),7.61 (1H, dd, J=1.8 , 8.2 Hz), 8.14 (1H, d, J=9 1.8 Hz).

Ethyl-4-iodobenzoate

To a suspension of 10 g (40.32 mmol) of 4-iodobenzoic acid in 100 mlabsolute ethanol was added 2 ml thionyl chloride and the mixture wasthen heated at reflux for 3 hours. Solvent was removed in vacuo and theresidue was dissolved in 100 ml ether. The ether solution was washedwith saturated NaHCO₃ and saturated NaCl solutions and dried (MgSO₄).Solvent was then removed in vacuo and the residue Kugelrohr distilled(100° C.; 0.55 mm) to give the title compound as a colorless oil, PMR(CDCl₃): δ1.42 (3H, t, J-7 Hz), 4,4 (2H, q, J-7 Hz), 7.8 (4H).

6-iodonicotinic acid

Sodium iodide (20.59 g, 137.40 mmol) was cooled to -78° C. under argonand then hydriodic acid (97.13 g, 759.34 mmol) was added. The coolingbath was removed and the suspension was stirred for 5 minutes. To thismixture was added 6-chloronicotinic acid (22.09 g, 140.20 mmol) and theresulting mixture was slowly warmed to ambient temperature withstirring. The mixture was heated to reflux at 125° C. for 24 hours,cooled to ambient temperature and poured into acetone (500 ml) at 0C.The yellow solid was collected by filtration and washed with 200 ml of1N aqueous NaHSO₃ solution. Recrystallization from methanol (crystalswere washed with ethyl ether) afforded the title compound as whitecrystals: mp 177°-179° C. lit. mp 187-192, Newkome et al. "ReductiveDehalogenation of Electron-Poor Heterocycles: Nicotinic AcidDerivatives" J Org. Chem. 51: 953-954 (1986). 1H NMR (DMSO-d₆): δ8.81(1H, dd, J=0.8, 2.4 Hz), 8.01 (1H, dd, J=0.8, 8.2 Hz), 7.91 (1H, dd,J=2.4, 8.2 Hz).

Ethyl 6-iodonicotinoate

To a suspension of 6-iodonicotinic acid (23.38 g, 94.20 mmol) indichloromethane (100 ml) was added a solution of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (19.86 g,103.6 mmol) in dichloromethane (250 ml). To this mixture was addedethanol (12.40 g, 269.27 mmol) followed by dimethylaminopyridine (1.15g, 9.41 mmol). The mixture was heated at 50° C. for 24.5 hours,concentrated in vacuo, and diluted with water (200 ml) then extractedwith ethyl ether (550 ml). The combined organic phases were washed withsaturated aqueous NaCl, dried (MgSO₄) and concentrated to a yellowsolid. Purification by flash chromatography (silica, 10% EtOAc-hexane)afforded the title compound as white needles: mp 48°-49° C.; 1H NMR(CDCl₃): δ8.94 (1H, d, J=2.1 Hz), 7.91 (1dd, J=2.1, 8.2 Hz), 7.85 (1H,d, J=8.2 Hz), 4.41 (2H, q, J=7.1 Hz), 1.41 (3H, t, J=7.1 Hz).

Ethyl 4-(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-naphthalenyl)ethynyl!benzoate(Compound F)

To a solution of 4 g (21.7 mmol) of7-ethynyl-3,4-dihydro-4,4-dimethylnaphthalen-1(2H)-one (Compound E )flushed for 15 minutes with a stream of argon, and 6 g (21.7 mmol) ofethyl 4-iodobenzoate in 100 ml of triethylamine was added 5 g (7.2 mmol)of bis(triphenylphosphine)palladium(II) chloride and 1.4 g (7.2 mmol) ofcuprous iodide. The mixture was flushed with argon for 5 minutes andthen stirred at ambient temperature for 18 hours. The reaction mixturewas filtered through Celite and the filtrate was concentrated in vacuo.Purification by flash chromatography (silica, 10% EtOAc-hexane) yieldedthe title compound as a white solid. PMR (CDCl₃) : δ1.41 (3H, t, J=7.2Hz), 1.41 (6H, s), 2.04 (2H, t, J=6.5 Hz), 2.76 (2H, t, J=6.5 Hz), 4.40(2H, q, J=7.2 Hz), 7.44 (1H, d, J=8.2 Hz), 7.59 (2H, d, J=8.4 Hz), 7.68(1H, dd, J=1.8, 8.2 Hz), 8.04 (2H, d, J=8.4 Hz), 8.15 (1H, d, J=1.8 Hz).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G)

To a cold solution (-78° C.) of 291.6 mg (1.59 mmol) of sodiumbis(trimethylsily)amide in 5.6 ml of THF was added a solution of 500.0mg (1.44 mmol) of ethyl 4-(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-naphthalenyl)ethynyl!benzoate(Compound F) in 4.0 ml of THF. The reaction mixture was stirred at -78°C. for 35 minutes and then a solution of 601.2 mg (1.59 mmol) of5-chloro(2-bis-triflouromethylsulfonyl)imide in 4.0 ml of THF was added.After stirring at -78° C. for 1 hour, the solution was warmed to 0° C.and stirred for 2 hours. The reaction was quenched by the addition ofsaturated aqueous NH₄ CI. The mixture was extracted with EtOAc (50 ml)and the combined organic layers were washed with 5% aqueous NaOH, water,and brine. The organic phase was dried over Na₂ SO₄ and thenconcentrated in vacuo to a yellow oil. Purification by columnchromatography (silica, 7% EtOAc-hexanes) yielded the title compound asa colorless solid. 1H NMR (CDCl₃): δ8.04 (2H, dd, J=1.8, 8.4 Hz), 7.60(2H, dd, J=1.8, 8.4 Hz), 7.51 (2H, m), 7.32 (1H, d, J=8.0 Hz), 4.40 (2H,q, J=7.1 Hz), 6.02 (1H, t, J=5.0 Hz), 2.44 (2H, d, J=5.0 Hz), 1.43 (3H,t, J=7.1 Hz), 1.33 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1)

A solution of 4-lithiotoluene was prepared by the addition of 189.9 mg(1.74 ml, 2.96 mmol) of t-butyl lithium (1.7M solution in hexanes) to acold solution (-78° C.) of 253.6 mg (1.482 mmol) of 4-bromotoluene in2.0 ml of THF. After stirring for 30 minutes a solution of 269.4 mg(1.977 mmol) of zinc chloride in 3.0 ml of THF was added. The resultingsolution was warmed to room temperature, stirred for 30 minutes, andadded via cannula to a solution of 472.9 mg (0.988 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) and 50 mg (0.04 mmol) oftetrakis(triphenylphosphine)palladium(0) in 4.0 ml of THF. The resultingsolution was heated at 50° C. for 45 minutes, cooled to room temperatureand diluted with sat. aqueous NH₄ Cl. The mixture was extracted withEtOAc (40 ml) and the combined organic layers were washed with water andbrine. The organic phase was dried over Na₂ SO₄ and concentrated invacuo to a yellow oil. Purification by column chromatography (silica, 5%EtOAc-hexanes) yielded the title compound as a colorless solid. 1H NMR(d6-acetone): δ1.35 (6H, s), 1.40 (3H, t, J=7.1 Hz), 2.36 (2H, d, J =4.7Hz), 2.42 (3H,s), 4.38 (2H, q, J=7.1 Hz), 5.99 (1H, t, J=4.7 Hz), 7.25(5H, m), 7.35 (2H, m), 7.52 (2H, d, J=8.5 Hz), 7.98 (2H, d, J=8.5 Hz).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethynyl!benzoate(Compound 1a)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate (Compound 1), 203.8 mg(0.43 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 58.2 mg (0.36 ml, 0.69 mmol) of phenyllithium (1.8M solution incyclohexane/Et₂ O), 116.1 mg (0.85 mmol) of zinc chloride and 13.8 mg(0.01 mmol) of tetrakis(triphenylphosphine)palladium(0). PMR (CDCl₃):δ1.36 (6H, s), 1.40 (3H, t, J=7.1Hz), 2.37 (2H, d, J=4.7 Hz), 4.38 (2H,q, J=7.1 Hz), 6.02 (1H, t, J=4.7 Hz), 7.20 (1H, d, J=1.5 Hz), 7.27 (1H,m), 7.39 (6H, m), 7.52 (2H, d, J=8.2 Hz), 7.98 (2H, d, J=8.2 Hz).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(3-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 2)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate (Compound 1), 250.0 mg(0.522 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 284.8 mg (2.090 mmol) of zinc chloride, 24 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and3-methylphenyl lithium (prepared by adding 201.2 mg (1.86 ml, 3.14 mmol)of tert-butyllithium (1.7M solution in pentane) to a cold solution (-78°C.) of 274.0 mg (1.568 mmol) of 3-methylbromobenzene in 2.0 ml of THF).1H NMR (CDCl₃): δ7.99 (2H, d, J=8.4 Hz), 7.51 (2H, d, J=8.4 Hz),7.39-7.14 (7H, m), 5.99 (1H, t, J=4.7 Hz), 4.37 (2H, q, J=7.1 Hz), 2.60(3H, s), 2.35 (2H, d, J=4.7 Hz), 1.39 (3H, t, J=7.1 Hz), 1.34 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 3)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1), 200.0 mg (0.418 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 199.4 mg (1.463 mmol) of zinc chloride, 24 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) in 4.0 ml of THF, and2-methylphenyl lithium (prepared by adding 133.9 mg (1.23 ml, 2.09 mmol)of tert-butyllithium (1.7M solution in pentane) to a cold solution (-78°C.) of 178.7 mg (1.045 mmol) of 2-methylbromobenzene in 2.0 ml of THF).1H NMR (CDCl₃): δ7.97 (2H, d, J=8.4 Hz), 7.50 (2H, d, J=8.4 Hz),7.49-7.19 (6H, m), 6.81 (1H, d, J=1.6 Hz), 5.89 (1H, t, J=4.5 Hz), 4.36(2H, q, J=7.1 Hz), 2.43-2.14 (2H, dq, J=3.7, 5.4 Hz), 2.15 (3H, s),1.39-1.34 (9H, m).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(3,5-dimethylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 4)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate (Compound 1), 250.0 mg(0.522 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 249.0 mg (1.827 mmol) of zinc chloride, 24 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and3,5-dimethylphenyl lithium (prepared by adding 167.7 mg (1.54 ml, 2.62mmol) of tert- butyllithium (1.7M solution in pentane) to a coldsolution (-78° C.) of 249.0 mg (1.305 mmol) of 3,5-dimethylbromobenzenein 2.0 ml of THF). 1H NMR (CDCl₃): δ7.98 (2H, d, J=8.4 Hz), 7.52 (2H, d,J=8.4 Hz), 7.40-7.33 (2H, m), 7.20 (1H, d, J=1.6 Hz), 7.00 (1H, s), 6.97(2H, s), 5.97 (1H, t, J=4.8 Hz), 4.37 (2H, q, J=7.1 Hz), 2.36 (6H, s),2.34 (2H, d, J=4.8 Hz), 1.39 (3H, t, J=7.1 Hz), 1.37 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-ethyl)-2-naphthalenyl)ethynyl!benzoate(Compound 5)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate (Compound 1), 250.0 mg(0.522 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 249.0 mg (1.827 mmol) of zinc chloride, 24 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and4-ethylphenyl lithium (prepared by adding 167.7 mg (1.54 ml, 2.62 mmol)of tert-butyllithium (1.7M solution in pentane) to a cold solution (}3178° C.) of 244.0 mg (1.305 mmol) of 4-ethylbromobenzene in 2.0 ml ofTHF). 1H NMR (CDCl₃): δ7.99 (2H, d, J=8.4 Hz), 7.51 (2H, d, J=8.4 Hz),7.42-7.24 (7H, m) 5.99 (1H, t, J=4.7 Hz), 4.37 (2H, q, J=7.1 Hz), 2.71(2H, q, J=7.6 Hz), 2.35 (2H, d, J=4.7 Hz), 1.39 (3H, t, J=7.1 Hz), 1.34(6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-(1,1-dimethylethyl)phenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 6)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1), 250.0 mg (0.52 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 142.4 mg (1.045 mmol) of zinc chloride and 4-tert-butylphenyllithium (prepared by adding 100.6 mg (0.97 ml, 1.57 mmol) oftert-butyllithium (1.5M solution in pentane) to a cold solution (-78°C.) of 167.0 mg (0.78 mmol) of 4-tert-butylbromobenzene in 1.0 ml ofTHF) . 1H NMR (CDCl₃): δ7.99 (2H, d, J 8.4 Hz), 7.55 (2H, d, J=8.4 Hz),7.28-7.45 (7H, m), 6.02 (1H, t, J=4.9 Hz), 4.38 (2H, q, J=7.2 Hz), 2.36(2H, d, J=4.9 Hz), 1.59 (3H, s), 1.40 (3H, t, J=7.2 Hz), 1.39 (9H, s),1.35 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-chlorophenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 7)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1), 250.0 mg (0.522 mmol) of ethyl 4- (5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 249.0 mg (1.827 mmol) of zinc chloride, 24 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and4-chlorophenyl lithium (prepared by adding 167.7 mg (1.54 ml, 2.62 mmol)of tert-butyllithium (1.7M solution in pentane) to a cold solution (-78°C.) of 252.4 mg (1.305 mmol) of 4-chloro-1-bromobenzene in 2.0 ml ofTHF). 1H NMR (CDCl₃): δ7.98 (2H, d, J=8.4 Hz), 7.53 (2H, d, J=8.4 Hz),7.40-7.27 (6H, m), 7.12 (1H, d, J=1.6 Hz), 6.00 (1H, t, J=4.8 Hz), 4.37(2H, q, J=7.1 Hz), 2.35 (2H, d, J=4.8 Hz), 1.40 (2H, t, J=7.1 Hz), 1.34(6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl-2-naphthalenyl)ethynyl!benzoate(Compound 8)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate (Compound 1), 250.0 mg(0.522 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 249.0 mg (1.827 mmol) of zinc chloride, 24 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and4-methoxyphenyl lithium (prepared by adding 167.7 mg (1.54 ml, 2.62mmol) of tert-butyllithium (1.7M solution in pentane) to a cold solution(-78° C.) of 244.1 mg (1.305 mmol) of 4-methoxy-1-bromobenzene in 2.0 mlof THF). 1H NMR (CDCl₃): δ7.98 (2H, d, J=8.5 Hz), 7.52 (2H, d, J=8.6Hz), 7.40-7.21 (5H, m), 6.95 (2H, d, J=8.7 Hz), 5.97 (1H, t, J=4.7 Hz),4.37 (2H, q, J=7.1 Hz), 4.34 (3H, s), 2.34 (2H, d, J=4.7 Hz), 1.39 (3H,t, J=7.1 Hz), 1.34 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-trifluoromethylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 9)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate (Compound 1), 250.0 mg(0.522 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 249.0 mg (1.827 mmol) of zinc chloride, 24 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and4-trifluoromethylphenyl lithium (prepared by adding 167.7 mg (1.54 ml,2.62 mmol) of tert-butyllithium (1.7M solution in pentane) to a coldsolution (-78° C.) of 296.6 mg (1.305 mmol) of 4-trifluoromethylbromobenzene in 2.0 ml of THF). 1H NMR (CDCl₃): δ7.98(2H, d, J=8.5 Hz), 7.67 (2H, d, J=8.3 Hz), 7.54-7.36 (6H, m), 7.10 (1H,d, J=1.6 Hz), 6.06 (1H, t, J=4.8 Hz), 4.37 (2H, q, J=7.1 Hz), 2.38 (2H,d, J=4.8 Hz), 1.39 (3H, t, J=7.1 Hz), 1.35 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-pyridyl)-2-naphthalenyl)ethynyl!benzoate(Compound 10)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1), 250.0 mg (0.52 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 142.4 mg (1.045 mmol) of zinc chloride and 2-lithiopyridine(prepared by the addition of 100.6 mg (0.97 ml, 1.57 mmol) oftert-butyllithium (1.5M solution in pentane) to a cold solution (-78°C.) of 123.8 mg (0.784 mmol) of 2-bromopyridine in 1.0 ml of THF). 1HNMR (d6-acetone): δ8.64 (1H, m), 7.99 (2H, d, J=8.5 Hz), 7.85 (1H, ddd,J=1.8, 7.7, 9.5 Hz), 7.58 (2H, d, J=8.4 Hz), 7.50 (1H, d, J=7.7 Hz),7.47 (2 H, d, J=1.1 Hz), 7.35 (2H, m), 6.32 (1H, t, J=4.8 Hz), 4.34 (2H,q, J=7.2 Hz), 2.42 (2H, d, J=7.4 Hz), 1.35 (3H, t, J=7.0 hz), 1.35 (6H,s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(3-pyridyl)-2-naphthalenyl)ethynyl!benzoate(Compound 11)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate (Compound 1), 170.0 mg(0.35 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate (Compound G) was converted into the titlecompound (colorless solid) using 142.4 mg (1.045 mmol) of zinc chlorideand 3-lithiopyridine (prepared by the addition of 100.2 mg (0.92 ml,1.56 mmol) of tert-butyllithium (1.5M solution in pentane) to a coldsolution (-78° C.) of 123.8 mg (0.784 mmol) of 3-bromopyridine in 1.0 mlof THF). 1H NMR (CDCl₃): δ8.63-8.61 (2H, dd, J=1.7 Hz), 7.99 2H, d,J=8.4 Hz), 7.67 (1H, dt, J=7.9 Hz), 7.52 (2H, d, J=8.4 Hz), 7.43-7.34(3H, m), 7.10 (1H, d, J=1.6 Hz), 6.07 (1H, t, J =4.7 Hz), 4.37 (2H, q,J=7.1 Hz), 2.40 (2H, d, J=4.7 Hz), 1.390 (3H, t, J=7.1 Hz), 1.36 (6H,s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-methyl-5-pyridyl)-2-naphthalenyl)ethynyl!benzoate(Compound 12)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenlyl)-2-naphthalenyl)ethynyl!benzoate (Compound1), 250.0 mg (0.522 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 142.4 mg (1.045 mmol) of zinc chloride and2-methyl-5-lithiopyridine (prepared by the addition of 100.5 mg (0.92ml, 1.57 mmol) of tert-butyllithium (1.7M solution in pentane) to a coldsolution (-78° C.) of 134.8 mg (0.784 mmol) of 2-methyl-5-bromopyridinein 1.0 ml of THF). 1H NMR (CDCl₃): δ8.50 (1H, d, J=2.2 Hz), 7.99 (2H, d,J=8.3 Hz), 7.56 (1H, dd, J=2.3, 8.0 Hz), 7.53 (2H, d, J=8.4 Hz), 7.43(1H, dd, J=2.3, 8.0 Hz), 7.37 (2H, d, J=8.0 Hz), 7.21 (1H, d, J=8.1 Hz),7.11 (1H, d, J=1.5 Hz), 6.04 (1H, t, J=4.7 Hz), 4.38 (2H, q, J=7.2 Hz),2.63 (3H, s), 2.38 (2H, d, J=4.6 Hz), 1.40 (3H, t, J=7.1 Hz), 1.35 (6H,s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(3-((2,2-dimethylethyl-dimethylsiloxy)phenyl)-2-naphthalenylyethynyl!benzoate (Compound H)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate (Compound G), 150.0 mg(0.314 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 150.0 mg (1.10 mmol) of zinc chloride, 24 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and3-((2,2-dimethylethyl)dimethylsiloxy)phenyl lithium (prepared by adding100.2 mg (0.92 ml, 1.564 mmol) of tert-butyllithium (1.7M solution inpentane) to a cold solution (-78° C.) of 226.0 mg (0.787 mmol) of3-((2,2-dimethylethyl)dimethylsiloxy)bromobenzene in 2.0 ml of THF). 1HNMR (CDCl₃): δ7.98 (2H, d, J=8.4 Hz), 7.51 (2H, d, J=8.4 Hz), 7.40-7.22(4H, m), 6.95 (1H, d, J=7.6 Hz), 6.84-6.82 (2H, m), 6.00 (1H, t, J=4.7Hz), 4.37 (2H, q, J=7.1 Hz), 2.35 (2H, d, J=4.7 Hz), 1.39 (3H, t, J=7.1Hz), 1.34 (3H, s), 0.99 (9H, s), 0.23 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-((2,2-dimethylethyl)-dimethylsiloxy)phenyl)-2-naphthalenyl)ethynyl!benzoate(Compound I)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1), 210.0 mg (0.439 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 209.0 mg (1.53 mmol) of zinc chloride, 24 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and4-((2,2-dimethylethyl)dimethylsiloxy)phenyl lithium (prepared by adding140.3 mg (1.30 ml, 2.19 mmol) of tert-butyllithium (1.7M solution inpentane) to a cold solution (-78° C.) of 315.0 mg (1.09 mmol) of4-((2,2-dimethylethyl)dimethylsiloxy)bromobenzene in 2.0 ml of THF). 1HNMR (CDCl₃): δ7.98 (2H, d, J=8.4 Hz), 7.51 (2H, d, J=8.4 Hz), 7.39-7.25(3H, m), 7.21 (2H, d, J=8.5 Hz), 5.87 (2H, d, J=8.5 Hz), 5.96 (1H, t,J=4.7 Hz), 4.37 (2H, q, J=7.1 Hz), 2.33 (2H, d, J=4.7 Hz), 1.39 (3H, t,J=7.1 Hz), 1.33 (6H, s), 1.01 (9H, s), 0.25 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(3-hydroxyphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 13)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(3-((2,2-dimethylethyl)-dimethylsiloxy)-phenyl)-2-naphthalenyl)ethynyl!benzoate(Compound H) 60.0 mg (0.114 mmol) in 1.0 ml of THF at room temperaturewas added 91.5 mg (0.35 ml, 0.35 mmol) of tetrabutylamonium flouride (1Msolution in THF). After stirring overnight, the solution was dilutedwith EtOAc and washed with H₂ O and saturated aqueous NaCl, before beingdried over MgSO₄. Removal of the solvents under reduced pressure,followed by column chromatography (4: 1, Hexanes:EtOAc) afforded thetitle compound as a colorless solid. 1H NMR (CDCl₃): δ7.98 (2H, d, J=7.8Hz), 7.52 (2H, d, J=8.3 Hz), 7.39-7.21 (4H, m), 6.93 (1H, d, J=7.5 Hz),6.84 (1H, d, 7.1 Hz), 6.83 (1H, s), 6.01 1H, t, J=4.7 Hz), 4.91 (1H, s),4.39 (2H, q, J=7.1 Hz), 2.35 (2H, d, J=4.7 Hz), 1.39 (3H, t, J=7.1 Hz),1.34 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-hydroxy!phenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 14)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-((2,2-dimethylethyl)-dimethylsiloxy)phenyl)-2-naphthalenyl)ethynyl!benzoate(Compound I) 50.0 mg (0.095 mmol) in 1.0 ml of THF at room temperaturewas added 73.2 mg (0.29 ml, 0.29 mmol) of tetrabutylamonium fluoride (1Msolution in THF). After stirring overnight, the solution was dilutedwith EtOAc and washed with H₂ O and saturated aqueous NaCl, before beingdried over MgSO₄. Removal of the solvents under reduced pressure,followed by column chromatography (4:1, Hexanes:EtOAc) afforded thetitle compound as a colorless solid. 1H NMR (CDCl₃): δ7.98 (2H, d, J=8.2Hz), 7.52 (2H, d, J=8.3 Hz), 7.41-7.20 (5H, m), 6.88 (2H, d, J=8.4 Hz),5.96 (1H, t, J=4.5 Hz), 4.37 (2H, q, J=7.1 Hz), 2.34 (2H, d, J=4.5 Hz),1.39 (3H, t, J=7.1 Hz), 1.34 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(5-methylthiazol-2-yl)-2-naphthalenyl)ethynyl!benzoate(Compound 15)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1), 264.0 mg (0.552 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 150.0 mg (1.10 mmol) of zinc chloride, 14 mg (0.012 mmol) oftetrakis(triphenylphosphine)palladium(0) in 4.0 ml of THF, and5-methylthiazol-2-yl lithium (prepared by adding 53.2 mg (0.53 ml, 0.83mmol) of n- butyllithium (1.55M solution in hexanes) to a cold solution(-78° C.) of 82.0 mg (0.83 mmol) of 5-methylthiazole in 5.0 ml of THF).1H NMR (CDCl₃): δ7.99 (2H, d, J=7.8 Hz), 7.88 (1H, d, J=1.5 Hz), 7.55(2H, d, J=7.8 Hz), 7.54 (1H, s), 7.45 (1H, dd, J=1.5, 8.0 Hz), 7.35 (1H,d, J=7.9 Hz), 6.48 (1H, t, J=4.8 Hz), 4.38 (2H, q, J=7.1 Hz), 2.51 (3H,s), 2.38 (2H, d, J=4.8 Hz), 1.40 (3H, s), 1.32 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl!benzoate(Compound 15a)

A solution of 2-lithiothiazole was prepared by the addition of 41.2 mg(0.42 ml, 0.63 mmol) of n-butyl-lithium (1.5M solution in hexanes) to acold solution (-78° C.) of 53.4 mg (0.63 mmol) of thiazole in 1.0 ml ofTHF. The solution was stirred at for 30 minutes and then a solution of113.9 mg (0.84 mmol) of zinc chloride in 1.5 ml of THF was added. Theresulting solution was warmed to room temperature, stirred for 30minutes and then the organozinc was added via cannula to a solution of200.0 mg (0.42 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) and 12.4 mg (0.01 mmol) oftetrakis(triphenylphosphine)palladium(0) in 1.5 ml of THF. The resultingsolution was heated at 50° C. for 45 minutes, cooled to room temperatureand diluted with sat. aqueous NH₄ Cl. The mixture was extracted withEtOAc (40 ml) and the combined organic layers were washed with water andbrine. The organic phase was dried over Na₂ SO₄ and concentrated invacuo to a yellow oil. Purification by column chromatography (silica,20% EtOAc-hexanes) yielded the title compound as a colorless oil. PMR(CDCl₃): δ1.35 (6H, s), 1.40 (3H, t, J=7.1 Hz), 2.42 (2H, d, J=4.8 Hz),4.38 (2H, q, J=7.1 Hz), 6.57 (1H, t, J=4.8 Hz), 7.33 (1H, d, J=3.3 Hz),7.36 (1H, d, J=8.0 Hz), 7.46 (1H, dd, J=1.7 , 8.1 Hz), 7.55 (2H, d,J=8.4 Hz), 7.87 (1H, d, J=1.7 Hz), 7.92 (1H, d, J=3.3 Hz), 8.00 (2H, d,J=8.4 Hz).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylthiazol-2-yl)-2-naphthalenyl)ethynyl!benzoate(Compound 16)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate (Compound 1), 295.0 mg(0.617 mmol) of ethyl 4- (5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 168.0 mg (1.23 mmol) of zinc chloride, 16 mg (0.014 mmol) oftetrakis(triphenylphosphine)palladium(0) in 6.0 ml of THF, and4-methylthiazol-2-yl lithium (prepared by adding 59.6 mg (0.60 ml, 0.93mmol) of n- butyllithium (1.55M solution in hexanes) to a cold solution(-78° C.) of 92.0 mg (0.93 mmol) of 4-methylthiazole in 6.0 ml of THF).1H NMR (CDCl₃): δ8.00 (2H, d, J=8.4 Hz), 7.80 (1H, d, J=1.7 Hz), 7.55(2H, d, J=8.4 Hz), 7.45 (1H, dd, J=1.7, 8.0 Hz), 7.35 (1H, d, J=8.0 Hz),6.87 (1H, s), 6.52 (1H, t, J=4.7 Hz), 4.37 (2H, q, J=7.2 Hz), 2.54 (3H,s), 2.39 (2H, d, J=4.7 Hz), 1.40 (3H, t, J =7.2 Hz), 1.33 (3H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4,5-dimethylthiazol-2-yl)-2-naphthalenylethynyl!benzoate (Compound 17)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1), 200.0 mg (0.418 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 110.0 mg (0.84 mmol) of zinc chloride, 12 mg (0.011 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and4,5-dimethylthiazol-2-yl lithium (prepared by adding 40.2 mg (0.39 ml,0.63 mmol) of n- butyllithium (1.55M solution in hexanes) to a coldsolution (-78° C.) of 71.0 mg (0.63 mmol) of 4,5-dimethylthiazole in 2.0ml of THF). 1H NMR (CDCl₃): δ8.00 (2H, d, J=8.4 Hz), 7.82 (1H, d, J=1.7Hz), 7.54 (2H, d, J=8.4 Hz), 7.43 (1H, dd, J 1.7, 8.0 Hz), 7.33 91H, d,J=8.0 Hz), 6.45 (1H, t, J=4.9 Hz), 4.38 (2H, q, J=7.1 Hz), 2.41 (3H, s),2.40 (3H, s), 2.37 (2H, d, J=4.9 Hz), 1.40 (3H, t, J=7.1 Hz), 1.32 (6H,s).

4- (5,6-Dihydro-5,5-dimethyl-8-(2-methyl-5-pridyl)-2-naphthalenyl)ethynyl!benzoic acid (Compound 18)

A solution of 81.7 mg (0.194 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-methyl-5-pyridyl)-2-naphthalenyl)ethynyl!benzoate (Compound 12) and 40.7 mg (0.969 mmol) ofLiOH-H₂ O in 3 ml of THF/water (3:1, v/v), was stirred overnight at roomtemperature. The reaction was quenched by the addition of saturatedaqueous NH₄ Cl and extracted with EtOAc. The combined organic layerswere washed with water and brine, dried over Na₂ SO₄ and concentrated invacuo to give the title compound as a colorless solid. 1H NMR (d6-DMSO):δ8.41 (1H, d, J=1.9 Hz), 7.90 (2H, d, J=8.3 Hz), 7.63 (1H, dd, J=2.3,7.9 Hz), 7.59 (2H, d, J=8.3 Hz), 7.49 (2H, m), 7.33 (1H, d, J=7.8 Hz),6.95 (1H, s), 6.11 (1H, t, J=4.5 Hz), 2.52 (3H, s), 2.37 (2H, d, J=4.6Hz), 1.31 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(2-pyridyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 19)

A solution of 80.0 mg (0.196 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-pyridyl)-2-naphthalenyl)ethynyl!benzoate(Compound 10) and 20.6 mg (0.491 mmol) of LiOH-H₂ O in 3 ml of THF/water(3:1, v/v), was stirred overnight at room temperature. The reaction wasquenched by the addition of saturated aqueous NH₄ Cl and extracted withEtOAc. The combined organic layers were washed with water and brine,dried over Na₂ SO₄ and concentrated in vacuo to give the title compoundas a colorless solid. 1H NMR (d6-DMSO): δ8.64 (1H, m), 7.94 (2H, d,J=8.3 Hz), 7.87 (1H, dt, J=1.7, 7.8 Hz), 7.58 (2H, d, J=8.3 Hz), 7.50(1H, d, J=8.2 Hz), 7.47 (2H, s), 7.37 (1H, m), 7.25 (1H, s), 6.30 (1H,t, J=4.6 Hz), 2.39 (2H, d, J=4.6 Hz), 1.31 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(3-methylphenyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 20)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(3-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 2) 30.0 mg (0.071 mmol) in 3 ml of EtOH and 2 ml of THF wasadded 28.0 mg (0.70 mmol, 0.7 ml) of NaOH (1.0M aqueous solution). Thesolution was heated to 50° C. for 2 hours, cooled to room temperature,and acidified with 10% HCI. Extraction with EtOAc, followed by dryingover Na₂ SO₄, and removal of the solvents under reduced pressureafforded the title compound as a colorless solid. 1H NMR (DMSO): δ7.90(2H, d, J=8.5 Hz), 7.59 (2H, d, J=8.5 Hz), 7.46 (2H, s), 7.32-7.13 (4H,m), 7.10 (1H, s), 6.98 (1H, t, J=4.5 Hz), 2.34 (3H, s), 2.31 (2H, d,J=4.5 Hz), 1.30 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(4-ethylphenyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 21)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-ethylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 5) 47.0 mg (0.108 mmol) in 3 ml of EtOH and 2 ml of THF wasadded 28.0 mg (0.70 mmol, 0.7 ml) of NaOH (1.0M aqueous solution). Thesolution was heated to 50° C. for 2 hours, cooled to room temperature,and acidified with 10% HCl. Extraction with EtOAc, followed by dryingover Na₂ SO₄, and removal of the solvents under reduced pressureafforded the title compound as a colorless solid. 1H NMR (DMSO): δ7.90(2H, d, J=8.3 Hz), 7.59 (2H, d, J=8.3 Hz), 7.46 (2H, s), 7.29-7.21 (4H,m), 7.02 (1H, s), 6.01 (1H, t, J=4.5 Hz), 2.64 (2H, q, J=7.5 Hz), 2.33(2H, d, J=4.5 Hz), 1.29 (6H, s), 1.22 (3H, t, J =7.5 Hz).

4-(5,6-Dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 22)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methoxyphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 8) 80.0 mg (0.183 mmol) in 3 ml of EtOH and 2 ml of THF wasadded 40.0 mg (1.00 mmol, 1.0 ml) of NaOH (1.0M aqueous solution). Thesolution was heated to 50° C. for 2 hours, cooled to room temperature,and acidified with 10% HCl. Extraction with EtOAc, followed by dryingover Na₂ SO₄, and removal of the solvents under reduced pressureafforded the title compound as a colorless solid. 1H NMR (DMSO): δ7.90(2H, d, J=8.3 Hz), 7.60 (2H, d, J=8.3 Hz), 7.45 (2H, s), 7.24 (2H, d,J=8.6 Hz), 7.02-6.89 (3H, m), 5.98 (1H, t, J=4.4 Hz), 3.79 (3H, s), 2.31(2H, d, J=4.7 Hz), 1.29 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(4-trifluoromethylphenyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 23)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-trifluoromethylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 9) 70.0 mg (0.148 mmol) in 3 ml of EtOH and 2 ml of THF wasadded 60.0 mg (1.50 mmol, 1.50 ml) of NaOH (1.0M aqueous solution). Thesolution was heated to 50° C. for 2 hours, cooled to room temperature,and acidified with 10% HCl. Extraction with EtOAc, followed by dryingover Na₂ SO₄, and removal of the solvents under reduced pressureafforded the title compound as a colorless solid. 1H NMR (DMSO): δ7.90(2H, d, J=8.3 Hz), 7.80 (2H, d, J=8.1 Hz), 7.61-7.47 (6H, m), 6.97 (2H,s), 6.16 (1H, t, J=4.5 Hz), 2.37 (2H, d, J=4.6 Hz), 1.30 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(3,5-dimethylphenyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 24)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(3,5-dimethylphenyl)-2-naphthalenyl)ethynyl!-benzoate(Compound 4) 90.0 mg (0.207 mmol) in 3 ml of EtOH and 2 ml of THF wasadded 48.0 mg (1.20 mmol, 1.20 ml) of NaOH (1.0M aqueous solution). Thesolution was heated to 50° C. for 2 hours, cooled to room temperature,and acidified with 10% HCl. Extraction with EtOAc, followed by dryingover Na₂ SO₄, and removal of the solvents under reduced pressureafforded the title compound as a colorless solid. 1H NMR (DMSO): δ7.90(2H, d, J=8.2 Hz), 7.59 (2H, d, J=8.2 Hz), 7.45 (2H, s), 7.00 (1H, s),6.97 (1H, s), 5.97 (1H, t, J=4.5 Hz), 2.31 (2H, d, J=4.5 Hz), 2.30 (6H,s), 1.29 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(4-chlorophenyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 25)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-chlorophenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 7) 80.0 mg (0.181 mmol) in 3 ml of EtOH and 2 ml of THF wasadded 48.0 mg (1.20 mmol, 1.20 ml) of NaOH (1.0M aqueous solution). Thesolution was heated to 50° C. for 2 hours, cooled to room temperature,and acidified with 10% HCl. Extraction with EtOAc, followed by dryingover Na₂ SO₄, and removal of the solvents under reduced pressureafforded the title compound as a colorless solid. 1H NMR (DMSO): δ7.90(2H, d, J=8.3 Hz), 7.60 (2H, d, J=8.3 Hz), 7.51-7.48 (4H, m), 7.34 (2H,d, J=8.4 Hz), 6.97 (1H, s), 6.07 (1H, t, J=4.5 Hz), 2.34 (2H, d, J=4.6Hz), 1.29 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(3-pyridyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 26)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(3-pyridyl)-2-naphthalenyl)ethynyl!benzoate(Compound 11) 45.0 mg (0.110 mmol) in 3 ml of EtOH and 2 ml of THF wasadded 48.0 mg (1.20 mmol, 1.20 ml) of NaOH (1.0M aqueous solution). Thesolution was heated to 50° C. for 2 hours, cooled to room temperature,and acidified with 10% HCl. Extraction with EtOAc, followed by dryingover Na₂ SO₄, and removal of the solvents under reduced pressureafforded the title compound as a colorless solid. 1H NMR (DMSO): δ 8.60(1H, d, J=4.6 Hz), 8.55 (1H, s), 7.90 (2H, d, J=8.3 Hz), 7.76 (1H, d,J=7.5 Hz), 7.60 (2H, d, J=8.3 Hz), 7.51-7.46 (3H, m), 6.94 (1H, s), 6.14(1H, t, J=4.5 Hz), 2.37 (2H, d, J=4.5 Hz), 1.31 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(2-methylphenyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 27)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-methylphenyl)-2-naphthalenyl)ethynyl!-benzoate(Compound 3) 80.0 mg (0.190 mmol) in 3 ml of EtOH and 2 ml of THF wasadded 60.0 mg (1.50 mmol, 1.50 ml) of NaOH (1.0M aqueous solution). Thesolution was heated to 50° C. for 2 hours, cooled to room temperature,and acidified with 10% HCl. Extraction with EtOAc, followed by dryingover Na₂ SO₄, and removal of the solvents under reduced pressureafforded the title compound as a colorless solid. 1H NMR (DMSO): δ7.89(2H, d, J=8.4 Hz), 7.57 (2H, d, J=8.4 Hz), 7.46 (2H, s), 7.29-7.14(4H, m), 6.59 (1H, s), 5.90 (1H, t, J=4.7 Hz), 2.39 (2H, m), 2.60 (3H,s), 1.39 (3H, s), 1.29 (3H, s).

4- (5,6-Dihydro-5,5-dimethyl-8-(3hydroxyphenyl)-2-naphthalenyl)ethynyl!benzoic acid (Compound 28)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(3-((2,2-dimethylethyl)-dimethylsiloxy)phenyl)-2-naphthalenyl)ethynyl!benzoate(Compound H) 40.0 mg (0.076 mmol) in 3 ml of EtOH and 2 ml of THF wasadded 40.0 mg (1.00 mmol, 1.00 ml) of NaOH (1.0M aqueous solution). Thesolution was heated to 50° C. for 2 hours, cooled to room temperature,and acidified with 10% HCl. Extraction with EtOAc, followed by dryingover Na₂ SO₄, and removal of the solvents under reduced pressureafforded the title compound as a colorless solid. 1H NMR (d6-acetone): δ7.90 (2H, d, J=8.3 Hz), 7.49 (2H, d, J=8.4 Hz), 7.35 (2H, s), 7.15-7.07(2H, m), 6.77-6.69 (3H, m), 5.92 (1H, t, J=4.7 Hz), 2.25 (2H, d, J=4.7Hz), 1.23 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(4-hydroxyphenyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 29)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-((2,2-dimethylethyl)-dimethylsiloxy)phenyl)-2-naphthalenyl)ethynyl!benzoate(Compound I)75.0 mg (0.143 mmol) in 3 ml of EtOH and 2 ml of THF wasadded 60.0 mg (1.50 mmol, 1.50 ml) of NaOH (1.0M aqueous solution). Thesolution was heated to 50° C. for 2 hours, cooled to room temperature,and acidified with 10% HCl. Extraction with EtOAc, followed by dryingover Na₂ SO₄, and removal of the solvents under reduced pressureafforded the title compound as a colorless solid. 1H NMR (d6-acetone): δ8.01 (2H, d, J=8.3 Hz), 7.59 (2H, d, J=8.4 Hz), 7.45 (2H, s), 7.20-7.17(3H, m), 6.92-6.89 (2H, m), 5.97 (1H, t, J=4.7 Hz), 2.35 (2H, d, J=4.7Hz), 1.34 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(5-methylthiazol-2-yl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 30)

To a solution of ethyl 4-5,6-dihydro-5,5-dimethyl-8-(5-methylthiazol-2-yl)-2-naphthalenyl!ethynylbenzoate(Compound 15) (100 mg, 0.23 mmol) and 4 ml of EtOH at room temperaturewas added aqueous NaOH (1 ml, 1M, 1 mmol). The resulting solution waswarmed to 50° C. for 1 hour and concentrated in vacuo. The residue wassuspended in a solution of CH₂ Cl₂ and ether (5:1) and acidified to pH 5with 1M aqueous HCl. The layers were separated and the organic layer waswashed with brine, dried (Na₂ SO₄), filtered and the solvents removedunder reduced pressure to give the title compound as a white solid. 1HNMR (d6-DMSO): δ 7.96 (1H, d, J=1.7 Hz), 7.95 (2H, d, J=8.0 Hz), 7.65(2H, d, J=8.0 Hz), 7.64 (1H, s), 7.53 (1H, dd, J=1.7, 8.0 Hz), 7.46 (1H,d, J=8.0 Hz), 6.59 (1H, t, J=4.5 Hz), 2.50 (3H, s), 2.39 (2H, d, J=4.5Hz), 1.27 (6H, s).

4-(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 30a)

A solution of 33.9 mg (0.08 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl!benzoate(Compound 15a) and 8.5 mg (0.20 mmol) of LiOH-H₂ O in 3 ml of THF/water(3:1, v/v), was stirred overnight at room temperature. The reaction wasquenched by the addition of sat. aqueous NH₄ Cl and extracted withEtOAc. The combined organic layers were washed with water and brine,dried over Na₂ SO₄ and concentrated in vacuo to give the title compoundas a colorless solid. PMR (d₆ -DMSO): δ 1.29 (6H, s), 2.42 (2H, d, J=4.6Hz), 6.68 (1H, t, J=4.6 Hz), 7.51 (2H, m), 7.62 (2H, d, J=8.2 Hz), 7.77(1H, d, J=3.3 Hz), 7.93 (2H, d, J=8.2 Hz), 7.98 (1H, d, J=3.3 Hz).

4-(5,6-Dihydro-5,5-dimethyl-8-(4-methylthiazol-2-yl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 31)

To a solution of ethyl 4-5,6-dihydro-5,5-dimethyl-8-(4-methylthiazol-2-yl)-2-naphthalenyl!ethynylbenzoate(Compound 16) (145.0 mg, 0.34 mmol) and 4 ml of EtOH at room temperaturewas added aqueous NaOH (1 ml, 1M, 1 mmol). The resulting solution waswarmed to 50° C. for 1 hour and concentrated in vacuo. The residue wassuspended in a solution of CH₂ Cl₂ and ether (5:1) and acidified to pH 5with 1M aqueous HCl. The layers were separated and the organic layer waswashed with brine, dried (Na₂ SO₄), filtered and the solvents removedunder reduced pressure to give the title compound as a white solid. 1HNMR (d6-DMSO): δ 7.94 (2H, d, J=8.1 Hz), 7.87 (1H, d, J=1.6 Hz), 7.63(2H, d, J=8.3 Hz), 7.50 (1H, dd, J=1.6, 8.1 Hz), 7.45 (1H, d, J=8.1 Hz),7.27 (1H, s), 6.58 (1H, t, J=4.8 Hz), 2.43 (3H, s), 2.37 (2H, d, J=4.8Hz), 1.26 (6H, s). ps 4-(5,6-Dihydro-5,5-dimethyl-8-(4,5-dimethylthiazol-2-yl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 32)

To a solution of ethyl 4-5,6-dihydro-5,5-dimethyl-8-(4,5-dimethylthiazol-2-yl)-2-naphthalenyl!ethynylbenzoate(Compound 17) (58.0 mg, 0.13 mmol) and 4 ml of EtOH at room temperaturewas added aqueous NaOH (1 ml, 1M, 1 mmol). The resulting solution waswarmed to 50° C. for 1 hour and concentrated in vacuo. The residue wassuspended in a solution of CH₂ Cl₂ and ether (5:1) and acidified to pH 5with 1M aqueous HCl. The layers were separated and the organic layer waswashed with brine, dried (Na₂ O₄), filtered and the solvents removedunder reduced pressure to give the title compound as a white solid. 1HNMR (d6-DMSO): δ 7.94 (2H, d, J=8.4 Hz), 7.86 (1H, d, J=1.6 Hz), 7.61(2H, d, J=8.3 Hz), 7.50 (1H, dd, J=1.6, 8.0 Hz), 7.45 (1H, d, J=8.0 Hz),6.51 (1H, t, J=4.9 Hz), 2.37 (3H, s), 2.36 (2H, d, J=4.6 Hz), 2.32 (3H,s), 1.26 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(5-methyl-2-thienyl)-2-naphthalenyl)ethynyl!benzoate (Compound 33)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1), 170.0 mg (0.366 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 202.0 mg (1.48 mmol) of zinc chloride, 24 mg (0.022 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF, and5-methyl-2-lithiothiophene (prepared by adding 58.6 mg (0.36 ml, 0.915mmol) of n-butyllithium (2.5M solution in hexanes) to a cold solution(-78° C.) of 89.8 mg (0.915 mmol) of 2-methylthiophene in 2.0 ml ofTHF). 1H NMR (CDCl₃): δ 8.00 (2H, d, J=8.3 Hz), 7.59 (1H, d, J=1.7 Hz),7.55 (2H, d, J=8.2 Hz), 7.43 (1H, dd, J=1.7, 8.0 Hz), 7.35 (1H, d, J=8.0Hz), 6.87 (1H, d, J=3.5 Hz), 6.74 (1H, d, J=2.8 Hz), 6.15 (1H, t, J=4.8Hz), 4.38 (2H, q, J=7.1 Hz), 2.52 (3H, s), 2.32 (2H, d, J=4.8 Hz). 1.40(3H, t, 7.1 Hz), 1.32 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-thienyl)-2-naphthalenyl)ethynyl!benzoate(Compound 33a)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1), 250.0 mg (0.52 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 186.8 mg (1.37 mmol) of zinc chloride 37.1 mg (0.03 mmol) oftetrakis(triphenylphosphine)palladium(0) and 2-lithiothiophene (preparedby the addition of 65.9 mg (0.69 ml, 1.03 mmol) of n-butyllithium (1.5Msolution in hexane) to a cold solution (-78° C.) of 86.5 mg (1.03 mmol)of thiophene in 1.0 ml of THF). PMR (CDCl₃): δ 1.33 (6H, s), 1.36 (3H,t, J=7.1 Hz), 2.38 (2H, d, J=4.7 Hz), 4.34 (2H, q, J=7.2 Hz), 6.25 (1H,t, J=4.7 Hz), 7.13 (2H, m), 7.47 (4H, m), 7.62 (2H, d, J=8.5 Hz), 8.00(2H, d, J=8.5 Hz).

4-(5,6-Dihydro-5,5-dimethyl-8-(5-methyl-2-thienol)-2-naphthalenyl)ethynyl!benzoicacid (Compound 34)

To a solution of ethyl 4-5,6-dihydro-5,5-dimethyl-8-(5-methyl-2-thienyl)-2-naphthalenyl!ethynylbenzoate(Compound 33) (35.0 mg, 0.082 mmol) in 2 ml of EtOH and 1 ml THF at roomtemperature was added aqueous NaOH (1 ml, 1M, 1 mmol). The resultingsolution was stirred at room temperature overnight and then acidifiedwith 10% HCl. Extraction with EtOAc, followed by drying over Na₂ SO₄,and removal of the solvents under reduced pressure afforded the titlecompound as a colorless solid. 1H NMR (d6-acetone): δ 8.03 (2H, d, J=8.6Hz), 7.63 (2H, d, J=8.6 Hz), 7.54-7.48 (3H, m), 6.89 (1H, m), 6.18 (1H,t, J=4.7 Hz), 2.49 (3H, s), 2.35 (2H, d, J=4.7 Hz), 1.32 (6H, s).

4-(5,6-dihydro-5,5-dimethyl-8-(2-thienyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 34a)

Employing the same general procedure as for the preparation of 4-(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl!benzoic acid (Compound 30a), 70.0 mg(0.17 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-thienyl)-2-naphthalenyl)ethynyl!benzoate(Compound 33a) was converted into the title compound (colorless solid)using 17.8 mg (0.42 mmol) of LiOH in H₂ O. PMR (d₆ -DMSO): δ 1.27 (6H,s), 2.33 (2H, d, J=4.9 Hz), 6.23 (1H, t, J=4.9 Hz), 7.14 (2H, m),7.38-7.56 (4H, m), 7.61 (2H, d, J=8.3 Hz), 7.92 (2H, d, J=8.3 Hz).

5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenecarboxylic acid(Compound K)

A solution of3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-7-bromonaphthalene (CompoundD) (250.0 mg, 0.764 mmol) in 2.0 ml of THF was cooled to -78° C. and 1.0ml of t-butyllithium (1.68 mmol, 1.7M solution in pentane) was addedslowly. After stirring for 1 hour at -78° C. gaseous CO₂ (generated byevaporation of Dry-Ice, and passed though a drying tube) was bubbledthrough the reaction for 1 hour. The solution was then allowed to warmto room temperature and the reaction was quenched by the addition of 10%HCl. Extraction with EtOAc was followed by washing the combined organiclayers with H₂ O and saturated aqueous NaCl, and drying over MgSO₄.Removal of the solvents under reduced pressure and washing of the solidwith hexanes afforded the title compound as a colorless solid. 1H NMR(CDCl₃): δ 7.94 (1H, dd, J=1.8, 8.1 Hz), 7.76 (1H, d, J=1.8 Hz), 7.45(1H, d, J=8.1 Hz), 7.24 (4H, m), 6.01 (1H, t, J=4.7 Hz), 2.40 (3H, s),2.36 (2H, d, J=4.7 Hz), 1.35 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonol!amino!-benzoate(Compound 35)

A solution of 170.0 mg (0.58 mmol)5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenecarboxylic acid(Compound K) 115.0 mg (0.70 mmol) of ethyl 4-aminobenzoate, 145.0 mg(0.76 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, and 92.4 mg (0.76 mmol) of 4-dimethylaminopyridine in 6.0ml of DMF was stirred overnight at room temperature. Ethyl acetate wasadded and the resulting solution washed with H₂ O, saturated aqueousNaHCO₃, and saturated aqueous NaCl, then dried over MgSO₄. After removalof the solvent under reduced pressure, the product was isolated as acolorless solid by column chromatography (10 to 15% EtOAc/hexanes). 1HNMR (CDCl₃): δ 8.02 (2H, d, J=8.7 Hz), 7.72 (2H, m), 7.65 (2H, d, J=8.7Hz), 7.52 (1H, d, J=1.8 Hz), 7.48 (1H, d, J=8.0 Hz), 7.25 (4H, m), 6.15(1H, t, J=4.9 Hz), 4.36 (2H, q, J=7.1 Hz), 2.40 (3H, s), 2.38 (2H, d,J=4.9 Hz), 1.39 (3H, t, J=7.1 Hz), 1.37 (6H), s).

4-(5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl!amino!-benzoicacid (Compound 36)

To a solution of 26.5 mg (0.06 mmol) ethyl 4(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl!amino!-benzoate(Compound 35) in 3.0 ml EtOH and 4.0 ml of THF was added 240.1 mg NaOH(6.00 mmol, 3.0 ml of a 2M aqueous solution). After stirring at roomtemperature for 72 hours, the reaction was quenched by the addition of10% HCl. Extraction with EtOAc, and drying of the organic layers overMgSO₄, provided a solid after removal of the solvent under reducedpressure. Crystallization from CH₃ CN afforded the title compound as acolorless solid. 1H NMR (d6-DMSO): δ 10.4 (1H, s), 7.91-7.81 (5H, m),7.54 (1H, d, J=8.1 Hz), 7.45 (1H, d, J=1.7 Hz), 7.23 (4H, s), 6.04 (1H,t, J=4.7 Hz) 2.35 (5H, s), 1.33 (6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methyl-phenyl)-2-naphthalenyl)carbonyl!oxy!-benzoate(Compound 37)

A solution of 25.0 mg (0.086 mmol)5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenecarboxylic acid(Compound K) 17.5 mg (0.103 mmol) of ethyl 4-hydroxybenzoate, 21.4 mg(0.112 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, and 12.6 mg (0.103 mmol) of 4-dimethylaminopyridine in2.0 ml of DMF was stirred overnight at room temperature. Ethyl acetatewas added and the resulting solution washed with H₂ O, saturated aqueousNaHCO₃, and saturated aqueous NaCl, before being dried over MgSO₄. Afterremoval of the solvent under reduced pressure, the product was isolatedby column chromatography as a pale-yellow solid (10% EtOAc/hexanes). 1HNMR (CDCl₃): δ 8.08 (2H, d, J=8.1 Hz), 8.05 (1H, dd, J=1.8, 8.1 Hz),7.89 (1H, d, J=1.8 Hz), 7.50 (2H, d, J=8.1 Hz), 7.22 (5H, m), 6.05 (1H,t, J=4.7 Hz), 4.37 (2H, q, J=7.1 Hz), 2.39 (2H, d, J=4.7 Hz), 2.38 (3H,s), 1.39 (3H, t, J=7.1 Hz), 1.37 (6H, s).

2-Trimethylsilylethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl!oxy!-benzoate(Compound 38)

A solution of 93.5 mg (0.320 mmol)5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenecarboxylic acid(Compound K) 76.0 mg (0.319 mmol) of2-trimethylsilylethyl-4-hydroxybenzoate, 80.0 mg (0.417 mmol) of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 51.0 mg(0.417 mmol) of 4-dimethylaminopyridine in 4.0 ml of DMF was stirredovernight at room temperature. Ethyl acetate was added and the resultingsolution washed with H₂ O, saturated aqueous NaHCO₃, and saturatedaqueous NaCl, before being dried over MgSO₄. After removal of thesolvent under reduced pressure, the product was isolated as a colorlesssolid by column chromatography (5% EtOAc/hexanes). 1H NMR (CDCl₃): δ8.08 (2H, d, J=8.8 Hz), 8.05 (1H, dd, J=1.8, 8.1 Hz), 7.50 (1H, d, J=8.1Hz), 7.26-7.18 (6H, m), 6.05 (1H, t, J=4,7 Hz), 4.42 (2H, t, J=8.4 Hz),2.40 (2H, d, J=4.7 Hz), 2.39 (3H, s), 1.38 (6H, s), 0.09 (9H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl!oxy!-benzoicacid (Compound 39)

A solution of 110.0 mg (0.213 mmol) 2-trimethylsilylethyl 4(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl!oxy!-benzoate(Compound 38) and 167.3 mg of tetrabutylammonium flouride (0.640 mmol,0.64 ml of a 1M solution in THF) in 2.0 ml THF was stirred at roomtemperature for 22 hours. Ethyl acetate was added and the resultingsolution washed with H₂ O and saturated aqueous NaCl then dried overMgSO₄. Removal of the solvents under reduced pressure and washing of theresidual solid with EtOAc and CH₃ CN afforded the title compound as acolorless solid. 1H NMR (d6-acetone): δ 8.10 (2H, d, J=8.8 Hz), 8.06(1H, dd, J=2.0, 8.1 Hz), 7.82 (1H, d, J=1.9 Hz), 7.64 (1H, d, J=8.1 Hz),7.35 (2H, d, J=8.6 Hz), 7.25 (4H, m), 6.08 (1H, t, J=4.7 Hz), 2.42 (2H,d, J=4.7 Hz), 2.35 (3H, s), 1.39 (6H, s).

Ethyl 2-fluoro-4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl!amino!-benzoate(Compound 40)

A solution of 115.0 mg (0.41 mmol)5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenecarboxylic acid(Compound K) 89.0 mg (0.49 mmol) of ethyl 2-fluoro-4-aminobenzoate,102.0 mg (0.53 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride, and 65.0 mg (0.53 mmol) of 4-dimethylaminopyridine in 5.0ml of DMF was stirred at 50° C. for 1 hour and then overnight at roomtemperature. Ethyl acetate was added and the resulting solution washedwith H₂ O, saturated aqueous NaHCO₃, and saturated aqueous NaCl, beforebeing dried over MgSO₄. After removal of the solvent under reducedpressure, the product was isolated as a colorless solid by columnchromatography (20% EtOAc/hexanes). 1H NMR (CDCl₃): δ 7.96 (1H, s), 7.89(1H, t, J=8.4 Hz), 7.70 (2H, m), 7.52 (1H, d, J=1.9 Hz), 7.45 (1H, d,J=8.1 Hz), 7.23 (5H, m), 6.04 (1H, t, J=4.8 Hz), 4.36 (2H, q, J=7.1 Hz),2.38 (3H, s), 2.35 (2H, d, J=4.8 Hz), 1.39 (3H, t, J=7.1 Hz), 1.36 (6H,s).

2-Fluoro-4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl!amino!-benzoicacid (Compound 41)

To a solution of 41.6 mg (0.091 mmol) ethyl 2- fluoro-4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl!amino!-benzoate(Compound 40) in 2.0 ml EtOH and 2.0 ml of THF was added 40.0 mg NaOH(1.00 mmol, 1.0 ml of a 1M aqueous solution). After stirring at roomtemperature for overnight, the reaction was quenched by the addition of10% HCl. Extraction with EtOAc, and drying of the organic layers overMgSO₄, provided a solid after removal of the solvent under reducedpressure. Crystallization from CH₃ CN afforded the title compound as apale-yellow solid. 1H NMR (d6-acetone): δ 9.84 (1H, s), 7.94-7.83 (3H,m), 7.64 (1H, dd, J=2.0 Hz), 7.53 (2H, d, J=8.1 Hz), 7.23 (4H, s), 6.04(1H, t, J=4.7 Hz), 2.38 (2H, d, J=4.7 Hz), 2.36 (3H, s), 1.35 (6H, s).

Ethyl 4(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)thiocarbonyl!amino!-benzoate(Compound 42)

A solution of 110.0 mg (0.25 mmol) ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl!amino!-benzoate(Compound 35) and 121.0 mg (0.30 mmol) of2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide!(Lawesson's Reagent) in 12.0 ml of benzene was refluxed overnight. Uponcooling to room temperature, the mixture was filtered and the filtrateconcentrated under reduced pressure. The title compound was isolated bycolumn chromatography (10 to 25% EtOAc/hexanes) as a yellow solid. 1HNMR (CDCl₃): δ 8.92 (1H, s), 8.06 (2H, t, J=8.5 Hz), 7.88-7.70 (3H, m),7.42 (2H, d, J=8.1 Hz), 7.18 (4H, m), 6.03 (1H, t, J=4.7 Hz), 4.37 (2H,q, J=7.1 Hz), 2.38 (3H, s), 2,36 (2H, d, J=4.7 Hz), 1.56 (3H, t, J=7.1Hz), 1.35 (6H, s).

4-(5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)thiocarbonyl!amino!-benzoicacid (Compound 43)

To a solution of 84.0 mg (0.184 mmol) ethyl 4(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)thiocarbonyl!amino!-benzoate(Compound 42) in 2.0 ml EtOH and 2.0 ml of THF was added 60.0 mg NaOH(1.50 mmol, 1.5 ml of a 1M aqueous solution). After stirring at roomtemperature overnight, the reaction was quenched by the addition of 10%HCl. Extraction with EtOAc, and drying of the organic layers over MgSO₄,provided a solid after removal of the solvent under reduced pressure.Crystallization from CH₃ CN afforded the title compound as a yellowsolid. 1H NMR (d6-acetone): δ 10.96 (1H, s), 8.05 (4H, m), 7.72 (1H, dd,J=2.0, 8.0 Hz), 7.54 (1H, s), 7.46 (1H, d, J=8.1 Hz), 7.20 (4H, m), 6.04(1H, t, J=4.7 Hz), 2.38 (2H, d, J=4.7 Hz), 2.33 (3H, s), 1.35 (6H, s).

2-acetyl-6-bromonaphthalene (Compound L)

To a cold (10° C.) mixture of 44.0 g (0.212 mol) of 2-bromonaphthaleneand 34.0 g (0.255 mol) of aluminum chloride in 400 ml of nitrobenzenewas added 21.0 g (267 mmol) of acetyl chloride. The mechanically stirredreaction mixture was warmed to room temperature, and heated to 40° C.for 18 hours. After cooling to 0° C. in an ice bath, the reaction wasquenched by the addition of 12M HCl (70 ml). The layers were separatedand the organic phase was washed with water and dilute aqueous Na₂ CO₃.Kugelrohr distillation, followed by recrystallization from 10%EtOAc-hexane yielded 23 g of the title compound as a tan solid. 1H NMR(CDCl₃): δ 8.44 (1H, br s), 8.04-8.10 (2H, m), 7.85 (1H, d, J=8.5 Hz),7.82 (1H, d, J=8.8 Hz), 7.64 (1H, d, J=8.8 Hz), 2.73 (3H, s).

6-bromo-2-naphthalenecarboxylic acid (Compound M)

To a solution of sodium hypochlorite (62 ml, 5.25% in water (w/w), 3.6g, 48.18 mmol) and sodium hydroxide (6.4 g, 160.6 mmol) in 50 ml ofwater was added a solution of 2-acetyl-6-bromonaphthalene (Compound L) 4g, (16.06 mmol) in 50 ml of 1,4-dioxane. The yellow solution was heatedto 70° C. in an oil bath for 2 hours, cooled to ambient temperature, andextracted with ethyl ether (2×50 ml). The aqueous layers were dilutedwith NaHSO₃ solution (until KI indicator solution remained colorless)and then acidified (pH<2) with 1N sulfuric acid to give a whiteprecipitate. The mixture was extracted with ethyl ether, and thecombined organic phase washed with saturated aqueous NaCl, dried (MgSO₄)and concentrated to give 3.54 g (88%) of the title compound as a solid.1H NMR (DMSO-d6): δ 8.63 (1H, br s), 8.32 (1H, d, J=2.0 Hz), 8.10 (1H,d, J=8.8 Hz), 8.00-8.05 (2H,m), 7.74 (1H, dd, J=2.0, 8.8 Hz).

Ethyl 6-bromo-2-naphthalenecarboxylate (Compound N)

To a solution of 6-bromo-2-naphthalenecarboxylic acid (Compound M) 3.1g, (12.43 mmol) in ethanol (30 ml, 23.55 g, 511.0 mmol) was added 18Msulfuric acid (2 ml). The solution was refluxed for 30 minutes, cooledto room temperature, and the reaction mixture partitioned betweenpentane (100 ml) and water (100 ml). The aqueous phase was extractedwith pentane (100 ml) and the combined organic layers washed withsaturated aqueous NaCl (100 ml), dried (MgSO₄), and concentrated toyield an off-white solid. Purification by flash chromatography (silica,10% EtOAc-hexane) afforded the title compound as a white solid. 1H NMR(CDCl₃): δ 8.58 (1H, br s), 8.10 (1H, dd, J=1.7, 9 Hz), 8.06 (1H, d, J=2Hz), 7.83 (1H, d, J=9 Hz), 7.80 (1H, d, J=9 Hz), 7.62 (1H, dd, J=2, 9Hz).

Ethyl (E)-4-2-(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-naphthalenyl)ethenyl!-benzoate(Compound O)

To a solution of 520.0 mg (2.00 mmol) of3,4-dihydro-4,4-dimethyl-7-bromo-1(2H)-naphthalenone (Compound B) and510.0 mg (2.90 mmol) of ethyl 4-vinylbenzoate in 4.0 ml of triethylamine(degassed by sparging with argon for 25 minutes), was added 124.0 mg(0.40 mmol) of tris(2-methylphenyl) phosphine, followed by 44.0 mg (0.20mmol) of palladium(II)acetate. The resulting solution was heated to 95°C. for 2.5 hours, cooled to room temperature, and concentrated underreduced pressure. Purification by column chromatography (10%EtOAc/hexanes) afforded the title compound as a colorless solid. 1H NMR(CDCl₃): δ 8.19 (1H, d, J=2.0 Hz), 8.03 (2H, d, J=8.4 Hz), 7.69 (1H, dd,J=2.0, 8.2 Hz), 7.57 (2H, d, J=8.4 Hz), 7.45 (1H, d, J=8.2 Hz), 7.20(2H, s), 4.39 (2H, q, J=7.1 Hz), 2.76 (2H, t, J=6.5 Hz), 2.04 (2H, t,J=6.5 Hz), 1.41(3H, t, J=7.1 Hz, and 6H, s).

Ethyl (E)-4-2-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethenyl!-benzoate(Compound P)

To a cold (-78° C.) solution of 440.0 mg (2.40 mmol) of sodiumbis(trimethylsilyl)amide in 10.0 ml of THF was added 700.0 mg (2.00mmol) of ethyl (E)-4-2-(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-naphthalenyl)ethenyl!-benzoate(Compound O) as a solution in 25.0 ml of THF. After stirring at -78° C.for 1.5 hours, 960.0 mg (2.40 mmol) of 2N,N-bis(trifluoromethylsulfonyl)amino!-5-chloropyridine was added in oneportion. After 30 minutes the solution was warmed to 0° C. and stirredfor 3 hours. The reaction was quenched by the addition of saturatedaqueous NH₄ Cl, and extracted with EtOAc. The combined extracts werewashed with 5% aqueous NaOH, dried (Na₂ SO₄), and the solvents removedunder reduced pressure. The title compound was isolated as a colorlesssolid by column chromatography (7% EtOAc/hexanes). 1H NMR (CDCl₃): δ8.04 (1H, d, J=8.4 Hz), 7.57 (2H, d, J=8.4 Hz), 7.52 (1H, s), 7.49 (1H,d, J=8.0 Hz), 7.33 (1H, d, J=8.0 Hz), 7.20 (1H, d, J=16.4 Hz), 7.10 (1H,d, J=16.4 Hz), 6.00 (1H, t, J=4.9 Hz), 4.39 (2H, q, J=7.1 Hz), 2.43 (2H,d, J=4.9 Hz), 1.41 (3H, t, J=7.1 Hz), 1.32 (6H, s).

Ethyl(E)-4-2-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethenyl!-benzoate(Compound 44)

A solution of 4-lithiotoluene was prepared at -78° C. by the addition of130.7 mg of t-butyllithium (2.04 mmol; 1.20 ml of a 1.7M solution inpentane) to a solution of 374.5 mg (2.20 mmol) of 4-bromotoluene in 2.5ml of THF. After 30 minutes a solution of 313.4 mg (2.30 mmol) of ZnCl₂in 2.0 ml of THF was added. The resulting solution was warmed to roomtemperature, stirred for 1.25 hour and then added via canula to asolution of 285.0 mg (0.590 mmol) of ethyl (E)-4-2-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethenyl!-benzoate(Compound P) and 29.0 mg (0.025 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF. The resultingsolution was stirred at room temperature for 1 hour and then at 55° C.for 2 hours. Upon cooling to room temperature the reaction was quenchedby the addition of saturated aqueous NH₄ Cl. The mixture was extractedwith EtOAc, and the combined extracts were washed with 5% aqueous NaOH,saturated aqueous NaCl, and dried over Na₂ SO₄ before being concentratedunder reduced pressure. The title compound was isolated by columnchromatography (10% EtOAC/hexanes) as a colorless solid. 1H NMR (CDCl₃):δ 7.96 (2H, d, J=8.1 Hz), 7.47 (2H, d, J=8.1 Hz), 7.43-7.16 (7H, ml,7.07 (1H, d, J=16.3 Hz), 6.93 (1H, d, J=16.3 Hz), 5.97 (1H, t, J=4.7Hz), 4.39 (2H, q, J=7.0 Hz), 2.41 (3H, s), 2.33 (1H, d, J=4.7 Hz), 1.38(3H, t, J=7.0 Hz), 1.33 (6H, s).

(E)-4-2-(5,6-Dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethenyl!-benzoicacid Compound 45

To a solution of 65.0 mg (0.190 mmol) of ethyl (E)-4-2-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethenyl!-benzoate(Compound 44) in 4.0 ml of THF was added 30.0 mg of LiOH (0.909 mmol,1.0 ml of a 1.1M solution) and 1.0 ml of MeOH. The solution was heatedto 55° C. for 3 hours, cooled to room temperature, and concentratedunder reduced pressure. The residue was dissolved in H₂ O and extractedwith hexanes. The aqueous layer was acidified to pH 1 with 10% HCl, andextracted with Et₂ O. The combined organic layers were washed withsaturated aqueous NaCl, diluted with EtOAc to give a clear solution, anddried over Na₂ SO₄. The solvents were removed under reduced pressure togive the title compound as a colorless solid. 1H NMR (d6-DMSO): δ 7.86(2H, d, J=8.4 Hz), 7.66 (2H, d, J=8.4 Hz), 7.58 (1H, dd, J=1.7, 8.1 Hz),7.41 (1H, d, J=8.1 Hz), 7.28 (1H, d, J=16.5 Hz), 7.23 (4H, s), 7.08 (1H,d, J=1.7 Hz), 7.07 (1H, d, J=16.5 Hz), 5.97 (1H, t, J=4.6 Hz), 2.35 (3H,s), 2.31 (1H, d, J=4.6 Hz), 1.29 (6H, s).

Ethyl 4- 2-(1,1-dimethyl-3-(4-methylphenyl)-5-indenyl)ethynyl!benzoate(Compound 47)

A solution of 32.0 mg (0.187 mmol) of 4-bromotoluene in 1.0 ml THF wascooled to -78° C. and 24.0 mg of t-butyllithium (0.375 mmol, 0.22 ml ofa 1.7M solution in pentane) was slowly added. The yellow solution wasstirred for 30 minutes at which time 29.8 mg (0.219 mmol) of ZnCl₂ wasadded as a solution in 1.0 ml THF. The resulting solution was warmed toroom temperature and after 30 minutes added to a second flask containing29.0 mg (0.062 mmol) of ethyl 4-2-(1,1-dimethyl-3-(trifluoromethylsulfonyl)oxy-5-indenyl)ethynyl!benzoate (Compound FF) and 2.9 mg (0.003 mmol) oftetrakis(triphenylphosphine)palladium (0) in 1.0 ml THF. The resultingsolution was warmed to 50° C. for 1 hour and then stirred at roomtemperature for 4 hours. The reaction was quenched by the addition ofsaturated aqueous NH₄ Cl, and then extracted with Et₂ O. The combinedorganic layers were washed with water, saturated aqueous NaCl, and driedover MgSO₄ before being concentrated under reduced pressure. The titlecompound was isolated as a colorless oil by column chromatography (10%Et₂ O/hexanes). 1H NMR (300 MHz, CDCl₃): δ 8.03 (2H, d, J=8.5 Hz), 7.66(1H, s), 7.58 (2H, d, J=8.5 Hz), 7.50 (2H, d, J=8.0 Hz), 7.46 (1H, d,J=7.9 Hz), 7.38 (1H, d, J=7.7 Hz), 7.28 (2H, d, J=9 Hz), 6.43 (1H, s),4.40 (2H, q, J=7.2 Hz), 2.43 (3H, s), 1.41 (3H, t; +6H, s).

4- 2-(1,1-dimethyl-3-(4-methylphenyl)-5-indenol)ethynyl!benzoic acid(Compound 48)

To a solution of 10.0 mg (0.025 mmol) of ethyl 4-2-(1,1-dimethyl-3-(4-methylphenyl)-5-indenyl)ethynyl!benzoate (Compound47) in 0.5 ml THF/H₂ O (3:1 v/v) was added 5.2 mg (0.12 mmol) LiOH H₂ O.After stirring at room temperature for 48 hours the solution wasextracted with hexanes and the aqueous layer was acidified withsaturated aqueous NH₄ Cl. Solid NaCl was added and the resulting mixtureextracted with EtOAc. The combined organic layers were dried (Na₂ SO₄)and concentrated under reduced pressure to give the title compound as acolorless solid. 1H NMR (300 MHz, d₆ -DMSO): δ 7.95 (2H, d, J=8.3 Hz),7.65 (2H, d, J=8.3 Hz), 7.57 (2H, m), 7.49 (3H, m), 7.30 (2H, d, J=7.9Hz), 6.61 (1H, s), 2.36 (3H, s), 1.36 (6H, s).

3-(4-bromothiophenoxy)propionic acid

To a solution of 1.44 g (35.7 mmol) of NaOH in 20.0 ml degassed H₂ O(sparged with argon) was added 6.79 g (35.7 mmol) of 4-bromothiophenol.The resulting mixture was stirred at room temperature for 30 minutes. Asecond flask was charged with 2.26 g (16.3 mmol) of K₂ CO₃ and 15 ml ofdegassed H₂ O. To this solution was added (in portions) 5.00 g (32.7mmol) of 3-bromopropionic acid. The resulting potassium carboxylatesolution was added to the sodium thiolate solution, and the resultingmixture stirred at room temperature for 48 hours. The mixture wasfiltered and the filtrate extracted with benzene, and the combinedorganic layers were discarded. The aqueous layer was acidified with 10%HCl and extracted with EtOAc. The combined organic layers were washedwith saturated aqueous NaCl, dried over MgSO₄, and concentrated underreduced pressure. The resulting solid was recrystallized from Et₂O--hexanes to give the title compound as off-white crystals. 1H NMR(CDCl₃): δ 7.43 (2H, d, J=8.4 Hz), 7.25 (2H, d, J=8.4 Hz), 3.15 (2H, t,J=7.3 Hz), 2.68 (2H, t, J=7.3 Hz).

2,3-dihydro-6-bromo-(4H)-1-benzothiopyran-4-one

A solution of 3.63 g (13.9 mmol) of 3-(4-bromothiophenoxy)propionic acidin 60 ml methanesulfonic acid was heated to 75° C. for 1.5 hours. Aftercooling to room temperature the solution was diluted with H₂ O andextracted with EtOAc. The combined organic layers were washed with 2Naqueous NaOH, H₂ O, and saturated aqueous NaCl and then dried overMgSO₄. Removal of the solvent under reduced pressure afforded a yellowsolid from which the product was isolated by column chromatography (3%EtOAc--hexanes) as a pale-yellow solid. 1H NMR (CDCl₃): δ 8.22 (1H, d,J=2.1 Hz), 7.48 1H, dd, J=2.1,8.3 Hz), 7.17 (1H, d, J=8.5 Hz), 3.24 (2H,t, J=6.4 Hz), 2.98 (2H, t, J=6.7 Hz).

2,3-dihydro-6-(2-trimethylsilylethynyl)-(4H)-1-benzothiopyran-4-one

A solution of 1.00 g (4.11 mmol)2,3-dihydro-6-bromo-(4H)-1-benzothiopyran-4-one and 78.3 mg (0.41 mmol)CuI in 15.0 ml THF and 6.0 ml Et₂ NH was sparged with argon for 5minutes. To this solution was added 2.0 ml (1.39 g, 14.2 mmol) of(trimethylsilyl)acetylene followed by 288.5 mg (0.41 mmol) ofbis(triphenylphosphine)palladium(II) chloride. The resulting darksolution was stirred at room temperature for 3 days and then filteredthrough a pad of Celite, which was washed with EtOAc. The filtrate waswashed with H₂ O and saturated aqueous NaCl before being dried overMgSO₄. The title compound was isolated as an orange oil by columnchromatography (4% EtOAc--hexanes). 1H NMR (CDCl₃): δ 8.13 (1H, d, J=1.9Hz), 7.36 (1H, dd, J=2.1, 8.2 Hz), 7.14 (1H, d, J=8.2 Hz), 3.19 (2H, d,J=6.3 Hz), 2.91 (2H, d, J=6.3 Hz), 0.21 (9H, s).

2,3-dihydro-6-ethenyl-(4H)- 1-benzothiopyran-4-one

A solution containing 600.0 mg (2.25 mmol) of2,3-dihydro-6-(2-trimethylsilylethynyl)-(4H)-1-benzothiopyran-4-one and100.0 mg (0.72 mmol) K₂ CO₃ in 15 ml MeOH was stirred at roomtemperature for 20 hours. The solution was diluted with H₂ O andextracted with Et₂ O. The combined organic layers were washed with H₂ Oand saturated aqueous NaCl before being dried over MgSO₄. Removal of thesolvents under reduced pressure afforded the title compound as an orangesolid. 1H NMR (CDCl₃): δ 8.17 (1H, d, J=1.8 Hz), 7.40 (1H, dd, J=1.8,8.2 Hz), 7.19 (1H, d, J=8.2 Hz), 3.22 (2H, t, J=6.3 Hz), 3.08 (1H, s)2.94 (2H, t, J=6.3 Hz).

Ethyl 4-2-(6-(2,3-dihydro-(4H)-1-benzothiopyran-4-onyl))ethynyl!benzoate

A solution of 405.0 mg (2.15 mmol)2,3-dihydro-6-ethynyl-(4H)-1-benzothiopyran-4-one and 594.0 mg (2.15mmol) of ethyl 4-iodobenzoate in 15 ml Et₃ N and 3 ml THF was spargedwith argon for 15 minutes. To this solution was added 503.0 mg (0.72mmol) of bis(triphenylphosphine)palladium(II) chloride and 137.0 mg(0.72 mmol) CuI. This solution was stirred for 20 hours at roomtemperature and then filtered through a pad of Celite, which was washedwith EtOAc. Removal of the solvents under reduced pressure afforded abrown solid. Column chromatography (3% EtOAc-hexanes) afforded the titlecompound as an orange solid. 1H NMR (d₆ -acetone): δ 8.15 (1H, d, J=2.0Hz), 8.02 (2H, d, J=8.5 Hz), 7.69 (2H, d, J=8.5 Hz), 7.61 (1H, dd,J=2.1, 8.3 Hz), 7.40 (1H, d, J=8.2 Hz), 4.35 (2H, q, J=7.1 Hz), 3.40(2H, t, J=6.3 Hz), 2.96 (2H, t, J=6.3 Hz), 1.37 (3H, t, J=7.1 Hz).

Ethyl 4-2-(6-(4-(trifluoromethylsulfonyl)oxy-(2H)-1-benzothiopyranyl))ethynyl!benzoate

To a solution of 221.9 mg (1.21 mmol) of sodium bis(trimethylsilyl)amidein 3.0 ml THF cooled to -78° C. was added 370.0 mg (1.10 mmol) of ethyl4- 2-(6-(2,3-dihydro-(4H)-1-benzothiopyran-4-onyl))ethynyl!benzoate in4.0 ml THF. After 30 minutes, a solution of 2-N,N-bis(trifluoromethylsulfonyl)amino!-5-chloropyridine in 4.0 ml THFwas slowly added. The reaction was slowly warmed to room temperature andafter 5 hours quenched by the addition of saturated aqueous NH₄ Cl. Themixture was extracted with EtOAc, and the combined organic layers werewashed with 5% aqueous NaOH, H₂ O, and saturated aqueous NaCl beforebeing dried over MgSO₄. Removal of the solvents under reduced pressure,followed by column chromatography (4% EtOAc-hexanes) afforded the titlecompound as a pale-yellow solid. 1H NMR (d₆ -acetone): δ 8.12 (2H, d,J=8.5 Hz), 7.66 (2H, d, J=8.5 Hz), 7.56 (1H, d, J=1.7 Hz), 7.49 (1H, dd,J=1.7, 8,1 Hz), 7.40 (1H, d, J=8.1 Hz), 6.33 (1H, t, J=5.7 Hz), 4.35(2H, q, J=7.1 Hz), 3.82 (2H, d, J=5.7 Hz), 1.37 (3H, t, J=7.1 Hz).

Ethyl 4-2-(6-(4-(4-methylphenyl)-(2H)-1-benzothiopyranyl))ethynyl!benzoate(Compound 49)

To a solution of 120.8 mg (0.70 mmol) of 4-bromotoluene in 2.0 ml THF at-78° C. was added 88.4 mg (1.38 mmol, 0.81 ml of a 1.7M solution inpentane) of t-butyllithium. After 30 minutes a solution of 131.6 mg(0.97 mmol) ZnCl₂ in 2.0 ml THF was added and the resulting pale-yellowsolution warmed to room temperature. Stirring for 40 minutes wasfollowed by addition of this solution to a second flask containing 129.2mg (0.28 mmol) of ethyl 4-2-(6-(4-(trifluoromethylsulfonyl)oxy-(2H)-1-benzothiopyranyl))ethynyl!benzoate,14.0 mg (0.012 mmol) tetrakis(triphenylphosphine)palladium (0), and 2.0ml THF. The resulting solution was heated to 50° C. for 5 hours, cooledto room temperature, and quenched by the addition of saturated aqueousNH₄ Cl. The mixture was extracted with EtOAc, and the combined organiclayers were washed with H₂ O and saturated aqueous NaCl, then dried(MgSO₄) and concentrated to an orange oil. The title compound wasisolated as a colorless solid by column chromatography (3 to 5%EtOAc-hexanes). 1H NMR (d₆ -acetone): δ 7.98 (2H, d, J=8.3 Hz), 7.58(2H, d, J=8.2 Hz), 7.44-7.38 (2H, m), 7.26-7.15 (5H, m), 6.14 (1H, t,J=5.8 Hz), 4.34 (2H, q, J=7.1 Hz), 3.53 (2H, d, J=5.8 Hz), 2.37 (2H, s),1.35 (3H, t, J=7.1 Hz).

4- 2-(6-(4-(4-methylphenyl)-(2H)-1-benzothiopyranyl))ethynyl!-benzoicacid (Compound 50)

To a solution of 29.0 mg (0.07 mmol) ethyl 4-2-(6-(4-(4-methylphenyl)-(2H)-1-benzothiopyranyl))ethynyl!benzoate(Compound 49) in 2.0 ml THF and 2.0 ml EtOH was added 160.0 mg (4.00mmol, 2.0 ml of a 2M aqueous solution). The resulting solution wasstirred at 35° C. for 2 hours, and then cooled to room temperature andstirred an additional 2 hours. The reaction was quenched by the additionof 10% aqueous HCl and extracted with EtOAc. The combined organic layerswere washed with H₂ O and saturated aqueous NaCl, and dried over Na₂SO₄. Removal of the solvents under reduced pressure afforded a solidwhich was washed with CH₃ CN and dried under high vacuum to give thetitle compound as a pale-yellow solid. 1H NMR (d₆ -DMSO): δ 7.90 (2H, d,J=8.4 Hz), 7.59 (2H, d, J=8.4 Hz), 7.40 (4H, m), 7.25-7.13 (4H, m), 7.02(1H, d, J=1.7 Hz), 6.11 (1H, t, J=5.7 Hz), 3.54 (2H, d, J=5.7 Hz), 2.34(3H, s).

3,4-Dihydro-4,4-dimethyl-7-acetyl-1(2H)-naphthalenone (Compound R); and3,4-dihydro-4,4-dimethyl-6-acetyl-1(2H)-naphthalenone (Compound S)

To a cold (0° C.) mixture of aluminum chloride (26.3 g, 199.0 mmols) indichloromethane (55 ml) was added acetylchloride (15 g, 192 mmols) and1,2,3,4-tetrahydro-1,1-dimethylnaphthalene (24.4 g, 152 mmols) indichloromethane (20 ml) over 20 minutes. The reaction mixture was warmedto ambient temperature and stirred for 4 hours. Ice (200 g) was added tothe reaction flask and the mixture diluted with ether (400 ml). Thelayers were separated and the organic phase washed with 10% HCl (50 ml),water (50 ml), 10% aqueous sodium bicarbonate, and saturated aqueousNaCl (50 ml) before being dried over MgSO₄. This solvent was removed bydistillation to afford a yellow oil which was dissolved in benzene (50ml).

To a cold (0° C.) solution of acetic acid (240 ml) and acetic anhydride(120 ml) was added chromiumtrioxide (50 g, 503 mmols) in small portionsover 20 minutes under argon. The mixture was stirred for 30 mins at 0°C. and diluted with benzene (120 ml). The benzene solution preparedabove was added with stirring via an addition funnel over 20 minutes.After 8 hours, the reaction was quenched by careful addition ofisopropanol (50 ml) at 0° C., followed by water (100 ml). After 15minutes, the reaction mixture was diluted with ether (1100 ml) and water(200 ml), and then neutralized with solid sodium bicarbonate (200 g).The ether layer was washed with water (100 ml), and saturated aqueousNaCl (2×100 ml), and dried over MgSO₄. Removal of the solvent underreduced pressure afforded a mixture of the isomeric diketones which wereseparated by chromatography (5% EtOAc/hexanes). (Compound R): 1H NMR(CDCl₃): δ 8.55 (1H, d, J=2.0 Hz), 8.13 (1H, dd, J=2.0, 8.3 Hz), 7.53(1H, d, J=8.3 Hz), 2.77 (2H, t, J=6.6 Hz), 2.62 (3H, s), 2.05 (2H, t,J=6.6 Hz), 1.41 (6H, s). (Compound S): 1H NMR (CDCl₃): δ 8.10 (1H, d,J=8.1 Hz), 8.02 (1H, d, J=1.6 Hz), 7.82 (1H, dd, J=1.6, 8.1 Hz), 2.77(2H, t, J=7.1 Hz), 2.64 (3H, s), 2.05 (2H, t, J=7.1 Hz), 1.44 (6H, s).

3,4-Dihydro-4,4-dimethyl-7-(2-(2-methyl-1,3-dioxolanyl))-1(2H)-naphthalenone(Compound T)

A mixture of 3,4-dihydro-4,4-dimethyl-7-acetyl-1(2H)-naphthalenone(Compound R) (140.0 mg, 0.60 mmol), ethylene glycol (55.0 mg, 0.90mmol), p-toluenesulfonic acid monohydrate (4 mg) and benzene (25 ml) wasrefluxed using a Dean-Stark apparatus for 12 hours. The reaction wasquenched by the addition of 10% aqueous sodium bicarbonate, andextracted with ether (2×75 ml). The combined organic layers were washedwith water (5 ml), and saturated aqueous NaCl (5 ml), and dried overMgSO₄. Removal of the solvent under reduced pressure afforded the titlecompound as an oil. 1H NMR (CDCl₃): δ 8.13 (1H, d, J=2.0 Hz), 7.64 (1H,dd, J=2.0, 8.2 Hz), 7.40 (1H, d, J=8.2 Hz), 3.97-4.10 (2H, m), 3.70-3.83(2H, m), 2.73 (2H, t, J=6.5 Hz), 2.01 (2H, t, J=6.5 Hz), 1.64 (3H, s),1.39 (6H, s).

1,2,3,4-Tetrahydro-1-hydroxy-1-(4-methylphenyl)-4,4-dimethyl-7-(2-(2-methyl-1,3-dioxolanyl))naphthalene(Compound U)

To a solution of 195.4 mg (1.00 mmol) p-tolulylmagnesiumbromide (1.0 ml;1M solution in ether) in 2 ml THF was added a solution of3,4-dihydro-4,4-dimethyl-7-(2-(2-methyl-1,3-dioxolanyl))-1(2H)-naphthalenone(Compound T) 135.0 mg, 0.52 mmol) in 5 ml THF. The solution was refluxedfor 16 hours, cooled to room temperature, and diluted with ether (50ml). The solution was washed with water (5 ml), saturated aqueous NH₄ Cl(5 ml), and dried over MgSO₄. Removal of the solvents under reducedpressure and column chromatography (5% EtOAc/hexanes) afforded the titlecompound as a solid. 1H NMR (CDCl₃): δ 7.37 (2H, d), 7.21 (1H, s), 7.13(2H, d, J=8.5 Hz), 7.08 (2H, d, J=8.5 Hz), 3.88-3.99 (2H, m), 3.58-3.75(2H, m), 2.34 (3H, s), 2.12-2.30 (2H, m), 1.79-1.90 (1H, m), 1.57 (3H,s), 1.48-1.58 (1H, m), 1.38 (3H, s), 1.31 (3H, s).

3,4-Dihydro-1-(4-methylphenyl)-4,4-dimethyl-7-acetylnaphthalene(Compound V)

A mixture of1,2,3,4-tetrahydro-1-hydroxy-1-(4-methylphenyl)-4,4-dimethyl-7-(2-(2-methyl-1,3-dioxolanyl))naphthalene(Compound U) 130.0 mg (0.38 mmol), p-toluenesulfonic acid monohydrate (4mg) and benzene (5 ml) was refluxed for 16 hours. Upon cooling to roomtemperature, the reaction mixture was diluted with ether (100 ml) andwashed with 10% aqueous sodium bicarbonate, water, and saturated aqueousNaCl. The organic layer was dried over MgSO₄ and the solvents wereremoved under reduced pressure to give the title compound as a solid. 1HNMR (CDCl₃): δ 7.83 (1H, dd, J=1.8,8.0 Hz), 7.66 (1H, d, J=1.8 Hz), 7.45(1H, d, J=8.0 Hz), 7.25 (2H, d, J=8.5 Hz), 7.22 (2H, d, J=8.5 Hz), 6.03(1H, t, J=6.3 Hz), 2.47 (3H, s), 2.41 (3H, s), 2.37 (2H, d, J=6.3 Hz),1.36 (6H, s).

(E)-3-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)-2-butenenitrile(Compound W)

To a slurry of NaH (48.0 mg, 2.00 mmol) in THF (6 ml), was addeddiethylcyanomethylphosphonate (450.0 mg, 2.50 mmol). After 40 mins, asolution of3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-7-acetylnaphthalene(Compound V) 95.0 mg, (0.33 mmol) in THF (4 ml) was added. The mixturewas stirred for 16 hours, diluted with ether (100 ml), and washed withwater, and saturated aqueous NaCl before being dried over MgSO₄. Removalof the solvents under reduced pressure, and column chromatography (3%EtOAc/hexanes) afforded the title compound as a solid. 1H NMR (CDCl₃): δ7.39 (1H, d, J=1H), 7.32 (1H, dd, J=2.0, 8.1 Hz), 7.20-7.25 (4H, brs),7.15 (1H, d, J=2.0 Hz), 6.03 (1H, t, J=6.0 Hz), 5.44 (1H, s), 2.42 (3H,s), 2.36 (2H, d, J=6.0 Hz), 2.35 (3H, s), 1.35 (6H, s).

(E)-3-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)-2-butanal(Compound X)

To a cold solution (-78° C.) of(E)-3-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)-2-butenenitrile(Compound W) 84.0 mg, 0.29 mmol) in dichloromethane (4 ml) was added0.50 ml (0.50 mmol) of diisobutylaluminumhydride (1M solution indichloromethane). After stirring for 1 hour, the reaction was quenchedat -78° C. by adding 2-propanol (1 ml) diluted with ether (100 ml). Uponwarming to room temperature, the solution was washed with water, 10%HCl, and saturated aqueous NaCl. The organic layer was dried over MgSO₄and the solvent removed under reduced pressure to give the titlecompound as an oil. 1H NMR (CDCl₃): δ 10.12 (1H, d, J=7.9 Hz), 7.43 (2H,s), 7.19-7.28 (5H, m), 6.27 (1H, d, J=7.9 Hz), 6.03 (1H, t, J=4.8 Hz),2.47 (3H, s), 2.42 (3H, s), 2.37 (2H, d, J=4.8 Hz), 1.37 (6H, s).

Ethyl(E,E,E)-3-methyl-7-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)-2,4,6-octatrienoate(Compound 51)

To a cold (-78° C.) solution ofdiethyl-(E)-3-ethoxycarbonyl-2-methylallylphosphonate prepared inaccordance with J. Org. Chem. 39: 821 (1974)! 264.0 mg, (1.00 mmol) inTHF (2 ml) was added 26.0 mg (0.41 mmol, 0.65 ml)of n-butyllithium inhexanes (1.6M solution) followed immediately by the addition of(E)-3-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalen-yl)-2-butenal(Compound X) 82.0 mg, 0.26 mmol) in THF (3 ml). After 1 hour, thereaction mixture was diluted with ether (60 ml), washed with water (5ml), saturated aqueous NaCl (5 ml) and dried over MgSO₄. After removalof the solvents under reduced pressure, the title compound was isolatedas an oil by column chromatography (5% EtOAc/hexanes, followed by HPLCusing 1% EtOAc/hexanes). 1H NMR (acetone-d6): δ 7.36-7.43 (2H, m),7.18-7.27 (4H, m), 7.17 (1H, d, J=1.7 Hz), 7.08 (1H, dd, J=11.2, 15.2Hz), 6.46 (1H, d, J=11.2 Hz), 6.38 (1H, d, J=15.2 Hz), 5.98 (1H, t,J=4.7 Hz), 5.78 (1H, s), 4.10 (2H, q, J=7.1 Hz), 2.35 (3H, s), 2.33 (3H,s), 2.32 (2H, d, J=4.7 Hz), 2.12 (3H, s), 1.31 (6H, s), 1.22 (3H, t,J=7.1 Hz).

(E,E,E)-3-methyl-7-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)-2,4,6-octatrienoicacid (Compound 52)

To a solution of ethyl(E,E,E)-3-methyl-7-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)-2,4,6-octatrienoate(Compound 51) 85.0 mg, 0.20 mmol) in THF (1 ml) and methanol (1 ml) wasadded 12.0 mg (0.50 mmol) of LiOH (0.5 ml, 1M solution). The mixture wasstirred for 6 hours, diluted with ether (60 ml), acidified with 10% HCl(1 ml). The solution was washed with water, and saturated aqueous NaCl,before being dried over MgSO₄. Removal of the solvents under reducedpressure afforded the title compound as a solid, which was purified byrecrystallization from acetone. 1H NMR (acetone-d6): δ 7.35-7.45 (2H,m), 7.19-7.28 (4H, m), 7.17 (1H, d, J=1.8 Hz), 7.09 (1H, dd, J=11.5,15.1 Hz), 6.48 (1H, d, J=11.5 Hz), 6.42 (1H, d, J=15.1 Hz),5.99 (1H, t,J=4.7 Hz),5.82 (1H, s), 2.36 (3H, s), 2.33 (2H, d, J=4.7 Hz), 2.32 (3H,s), 2.13 (3H, s), 1.32 (6H, s).

3,4-dihydro-4,4-dimethyl-7-nitro-1(2H)-naphthalenone (Compound Y)

To 1.7 ml (3.0 g, 30.6 mmol, 18M) H₂ SO₄ at -5° C. (ice-NaCl bath) wasslowly added 783.0 mg (4.49 mmol) of3,4-dihydro-4,4-dimethyl-1(2H)-naphthalenone. A solution of 426.7 mg(6.88 mmol, 0.43 ml, 16M) HNO₃, and 1.31 g (0.013 mol, 0.74 ml, 18M) H₂SO₄ was slowly added. After 20 minutes, ice was added and the resultingmixture extracted with EtOAc. The combined extracts were concentratedunder reduced pressure to give a residue from which the title compound,a pale yellow solid, was isolated by column chromatography (10%EtOAC/hexanes). 1H NMR (CDCl₃): δ 8.83 (1H, d, J=2.6 Hz), 8.31 (1H, dd,J=2.8, 8.9 Hz), 7.62 (1H, d, J=8.7 Hz), 2.81 (2H, t, J=6.5 Hz), 2.08(2H, t, J=6.5 Hz), 1.45 (6H, s).

3,4-dihydro-4,4-dimethyl-7-amino-1(2H)-naphthalenone (Compound Z) Asolution of 230.0 mg (1.05 mmol)3,4-dihydro-4,4-dimethyl-7-nitro-1(2H)-naphthalenone (Compound Y) in 5.0ml of EtOAc was stirred at room temperature with a catalytic amount of10% Pd-C under 1 atm of H₂ for 24 hours. The catalyst was removed byfiltration through a pad of Celite, and the filtrate concentrated underreduced pressure to give the title compound as a dark green oil. 1H NMR(CDCl₃): δ 7.30 (1H, d, J=2.7 Hz), 7.22 (1H, d, J=8.4 Hz), 6.88 (1H, dd,J=2.7, 8.5 Hz), 2.70 (2H, t, J=6.6 Hz), 1.97 (2H, t, J=6.6 HZ), 1.34(6H, s).

Ethyl 4-(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-naphthalenyl)azo!-benzoate(Compound AA)

To a solution of 198.7 mg (1.05 mmol)3,4-dihydro-4,4-dimethyl-7-amino-1(2H)-naphthalenone (Compound Z) in 5.0ml glacial acetic acid was added 180.0 mg (1.00 mmol) of ethyl4-nitrosobenzoate. The resulting solution was stirred overnight at roomtemperature, and then concentrated under reduced pressure. The productwas isolated from the residual oil as a red solid, by columnchromatography (15% EtOAc--hexanes). 1H NMR (CDCl₃): δ 8.57 (1H, d,J=2.0 Hz), 8.19 (2H, d, J=8.4 Hz), 8.07 (1H, d, J=8.0 Hz), 7.94 (2H, d,J=8.4 Hz), 7.58 (1H, d, J=8.6 Hz), 4.41 (2H, q, J=7.1 Hz), 2.79 (2H, t,J=6.6 Hz), 2.07 (2H, t, J=7.02 Hz), 1.44 (6H, s), 1.42 (3H, t, J=7.1Hz).

Ethel 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)azo!-benzoate(Compound BB)

To a solution of 90.4 mg sodium bis(trimethylsilyl)amide (0.48 mmol,0.48 ml of a 1.0M THF solution) in 2.0 ml THF at -78° C., was added153.0 mg (0.437 mmol) of ethyl 4-(5,6,7,8-tetrahydro-5,5-dimethyl-8-oxo-2-naphthalenyl)azo!-benzoate(Compound AA) in 2.0 ml THF. The dark red solution was stirred at -78°C. for 30 minutes and then 204.0 mg (0.520 mmol) of 2-N,N-bis(trifluoromethylsulfonyl)amino!-5-chloropyridine was added as asolution in 2.0 ml THF. The reaction mixture was allowed to warm to roomtemperature and after 3 hours it was quenched by the addition of H₂ O.The organic layer was concentrated to a red oil under reduced pressure.The product was isolated by column chromatography (25% EtOAc/hexanes) asa red oil. 1H NMR (CDCl₃): δ 8.21 (2H, d, J=8.6 Hz), 7.96 (2H, d, J=8.6Hz), 7.94 (2H, m), 7.49 (1H, d, J=8.2 Hz), 6.08 (1H, t, J=2.5 Hz), 4.42(2H, q, J=7.1 Hz), 2.49 (2H, d, J=4.8 Hz), 1.44 (3H, t, J=7.1 Hz), 1.38(6H, s).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)azo!-benzoate(Compound 46a)

A solution of 4-lithiotoluene was prepared by the addition of 62.9 mg(0.58 ml, 0.98 mmol) of t-butyl lithium (1.7M solution in pentane) to acold solution (-78° C.) of 84.0 mg (0.491 mmol) of 4-bromotoluene in 1.0ml of THF. After stirring for 30 minutes a solution of 107.0 mg (0.785mmol) of zinc chloride in 2.0 ml of THF was added. The resultingsolution was warmed to room temperature, stirred for 30 minutes, andadded via cannula to a solution of 94.7 mg (0.196 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)azo!-benzoate(Compound BB) and 25 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) in 2.0 ml of THF. The resultingsolution was heated at 50° C. for 1.5 hours, cooled to room temperatureand diluted with sat. aqueous NH₄ Cl. The mixture was extracted withEtOAc (40 ml) and the combined organic layers were washed with water andbrine. The organic phase was dried over Na₂ SO₄, concentrated in vacuo,and the title compound isolated as a red solid by column chromatography(25% EtOAc-hexanes) 1H NMR (CDCl₃): δ 8.21 (2H, d, J=8.6 Hz), 7.96 (2H,d, J=8.6 Hz), 7.94 (2H, m), 7.49 (1H, d, J 8.2 Hz), 6.08 (1H, t, J=2.5Hz), 4.42 (2H, q, J=7.1 Hz), 2.49 (2H, d, J=4.8 Hz), 1.44 (3H, t, J=7.1Hz), 1.38 (6H, s).

4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenal)azo!-benzoicacid (Compound 46b)

To a solution of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)azo!-benzoate(Compound 46a) 16.5 mg, 0.042 mmol) in THF (2 ml) and ethanol (1 ml) wasadded 80.0 mg (2.00 mmol) of NaOH (2.0 ml, 1M aqueous solution). Themixture was stirred for 12 hours at room temperature, acidified with 10%HCl, and extracted with EtOAc. The combined organic layers were washedwith water, and saturated aqueous NaCl, then dried over MgSO₄. Removalof the solvents under reduced pressure, and recrystallization of theresidue from EtOAC/hexane, afforded the title compound as a red solid.1H NMR (acetone-d6): δ 8.19 (2H, d, J=8.4 Hz), 7.92 (2H, d, J=8.5 Hz),7.88 (2H, dd, J=2.1, 6.1 Hz), 7.66 (1H, s), 7.64 (2H, d, J=2.3 Hz), 7.28(4H, d, J=3.0 Hz), 6.09 (1H, t, J=2.5 Hz), 2.42 (2H, d, J=4.8 Hz), 2.39(3H, s), 1.40 (6H, s).

6-(2-Trimethylsilyl)ethynyl-2,3-dihydro-3 3-dimethyl-1H-inden-1-one(Compound CC)

To a solution of 815.0 mg (3.41 mmol)6-bromo-2,3-dihydro-3,3-dimethyl-1H-inden-1-one (See Smith et al. Org.Prep. Proced. Int. 1978 10 123-131) in 100 ml of degassed Et₃ N (spargedwith argon for 20 min) was added 259.6 mg (1.363 mmol) of copper(I)iodide, 956.9 mg (1.363 mmol) ofbis(triphenylphosphine)palladium(II)chloride, and 3.14 g (34.08 mmol) of(trimethylsilyl)acetylene. This mixture was heated at 70° C. for 42hours, cooled to room temperature, and filtered through a pad of silicagel and washed with ether. The filtrate was washed with water, 1M HCl,water, and finally with saturated aqueous NaCl before being dried overMgSO₄. Concentration of the solution under reduced pressure, followed bycolumn chromatography (silica gel; 10% Et₂ O--hexanes) afforded thetitle compound as a brown oil. 1H NMR (300 MHz, CDCl₃): δ 7.79(1H, d,J=1.4 Hz), 7.69 (1H, dd, J=1.6, 8.3 Hz), 7.42 (1H, d, J=8.5 Hz), 2.60(2H, s), 1.41 (6H, s), 0.26 (9H, s).

6-Ethynyl-2,3-dihydro-3,3-dimethyl-1H-inden-1-one (Compound DD)

To a solution of 875.0 mg (3.41 mmol) 6-(2-trimethylsilyl)ethynyl-2,3-dihydro-3,3-dimethyl-1H-inden-1-one (CompoundCC) in 28 ml of MeOH, was added 197.3 mg (1.43 mmol) of K₂ CO₃ in oneportion. After stirring for 6 hours at room temperature the mixture wasfiltered though a pad of Celite and the filtrate concentrated underreduced pressure. The residual oil was placed on a silica gel column andeluted with 5% EtOAc-hexanes to give the title product as a colorlessoil. 1H NMR (300 MHz, CDCl3): δ 7.82 (1H, s), 7.72 (1H, dd, J=1.6, 7.8Hz), 7.47 (1H, d, J=8.4 Hz), 3.11 (1H, s), 2.61 (2H, s), 1.43 (6H, s).

Ethyl 4- 2-(5,6-dihydro-5,5-dimethyl-7-oxo-2-indenyl)ethynyl!benzoate(Compound EE)

A solution of 280.0 mg (1.520 mmol)6-ethynyl-2,3-dihydro-3,3-dimethyl-1H-inden-1-one (Compound DD) and419.6 mg (1.520 mmol) ethyl 4-iodobenzoate in 5 ml Et₃ N was spargedwith argon for 40 minutes. To this solution was added 271.0 mg (1.033mmol) of triphenylphosphine, 53.5 mg (0.281 mmol) of copper(I) iodide,and 53.5 mg (0.076 mmol) of bis(triphenylphosphine)palladium(II)chloride. The resulting mixture was heated to reflux for 2.5 hours,cooled to room temperature, and diluted with Et₂ O. After filtrationthrough a pad of Celite, the filtrate was washed with H₂ O, 1M HCl, H₂O, and saturated aqueous NaCl, then dried over MgSO₄, and concentratedunder reduced pressure. The title compound was isolated as a pale-yellowsolid by column chromatography (15% EtOAc-hexanes). 1H NMR (300 MHz,d6-acetone): δ 8.05 (2H, d, J=8.6 Hz), 7.87 (1H, dd, J=1.4, 8.1 Hz),7.75 (2H, m), 7.70 (2H, d, J=8.5 Hz), 4.36 (2H, q, J=7.1 Hz), 2.60 (2H,s), 1.45 (6H, s), 1.37 (3H, t, J=7.1 Hz).

Ethyl 4-2-(1,1-dimethyl-3-(trifluoromethyl-sulfonyl)oxy-5-indenyl)ethynyl!benzoate(Compound FF)

A solution of 88.0 mg (0.48 mmol) of sodium bis(trimethylsilyl)amide in0.5 ml THF was cooled to -78° C. and 145.0 mg (0.436 mmol) of ethyl 4-2-(5,6-dihydro-5,5-dimethyl-7-oxo-2-indenyl)ethynyl!benzoate (CompoundEE) was added as a solution in 1.0 ml THF. After 30 minutes 181.7 mg(0.480 mmol) of2-(N,N-bis(trifluoromethansulfonyl)amino)-5-chloro-pyridine was added asa solution in 1.0 ml THF. The reaction was allowed to slowly warm toroom temperature and quenched after 5 hours by the addition of saturatedaqueous NH₄ Cl. The mixture was extracted with EtOAc, and the combinedorganic layers washed with 5% aqueous NaOH, H₂ O, and saturated aqueousNaCl, then dried (MgSO₄) and concentrated under reduced pressure. Theproduct was isolated as a colorless solid by column chromatography (10%Et₂ O-hexanes). 1H NMR (300 MHz, d6-acetone): δ 8.05 (2H, d, J=8.3 Hz),7.69 (2H, d, J=8.4 Hz), 7.63 (2H, s), 7.55 (1H, s), 4.36 (2H, q, J=7.1Hz), 1.44 (6H, s), 1.37 (3H, t, J=7.1 Hz).

4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 60)

A solution of 142.6 mg (0.339 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1) and 35.6 mg (0.848 mmol) of LiOH-H₂ O in 12 ml of THF/water(4:1, v/v), was stirred overnight at room temperature. The reactionmixture was extracted with hexanes, and the hexane fraction extractedwith 5% aqueous NaOH. The aqueous layers were combined and acidifiedwith 1M HCl, and then extracted with EtOAc and Et₂ O. The combinedorganic layers were dried over Na₂ SO₄ and concentrated in vacuo to givethe title compound as a colorless solid. 1H NMR (d₆ -DMSO): δ 7.91 (2H,d, J=8.4 Hz), 7.60 (2H, d, J=8.4 Hz), 7.47 (2H, s), 7.23 (4H, q, J=8.1Hz), 7.01 (1H, s), 6.01 (1H, t, J=4.6 Hz), 2.35 (3H, s), 2.33 (2H, d,J=4.8 Hz), 1.30 (6H, s).

4- (5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethynyl!benzoicacid (Compound 60a)

Employing the same general procedure as for the preparation of 4-(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 30a), 27.0 mg (0.07 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethynyl!benzoate(Compound 1a) was converted into the title compound (colorless solid)using 5.9 mg (0.14 mmol) of LiOH in H₂ O. PMR (d₆ -DMSO): δ 1.31 (6H,s), 2.35 (2H, d, J=4.5 Hz), 6.05 (1H, t, J=J=J=4.5 Hz), 7.00 (1H, s),7.33 (2H, d, J=6.2 Hz), 7.44 (4H, m), 7.59 (2H, d, J=8.1 Hz), 7.90 (2H,d, J=8.1 Hz).

4-(5,6-Dihydro-5,5-dimethyl-8-(4-(1,1-dimethylethyl)phenyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 61)

A solution of 80.0 mg (0.173 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-(1,1-dimethylethyl)phenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 6) and 18.1 mg (0.432 mmol) of LiOH-H₂ O in 6 ml of THF/water(3:1, v/v), was stirred overnight at room temperature. The reactionmixture was extracted with hexanes, and the remaining aqueous layeracidified with 1M HCl, and then extracted with EtOAc. The combinedorganic layers were dried over Na₂ SO₄ and concentrated in vacuo to givethe title compound as a colorless solid. 1H NMR (d₆ -DMSO): δ 7.82 (2H,d, J=8.2 Hz), 7.44 (6H, m), 7.25 (2H, d, J=8.3 Hz), 7.02 (1H, s), 6.01(1H, t, J=4.6 Hz), 2.32 (2H, d, J=4.7 Hz), 1.32 (9H, s), 1.29 (6H, s).

Ethyl 2-fluoro-4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)thiocarbonyl!amino!-benzoate(Compound 62)

A solution of 54.4 mg (0.119 mmol) ethyl 2-fluoro-4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)carbonyl!amino!-benzoate(Compound 40) and 57.7 mg (0.143 mmol) of 2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide! (Lawesson'sReagent) in 12.0 ml of benzene was refluxed overnight. Upon cooling toroom temperature, the mixture was filtered and the filtrate concentratedunder reduced pressure. The title compound was isolated by columnchromatography (10 to 25% EtOAc/hexanes) as a yellow solid. 1H NMR(CDCl₃): δ 9.08 (1H, s), 7.92 (1H, br s), 7.90 (1H, t, J=8.2 Hz), 7.66(1H, dd, J=2.0, 6.0 Hz), 7.38 (3H, m), 7.18 (4H, m), 6.01 (1H, t, J=4.7Hz), 4.35 (2H, q, J=7.1 Hz), 2.36 (3H, s), 2.33 (2H, d, J=4.7 Hz), 1.38(3H, t, J=7.1 Hz), 1.33 (6H, s).

2-fluoro-4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)thiocarbonyl!amino!-benzoicacid (Compound 63)

To a solution of 46.5 mg (0.098 mmol) ethyl 2-fluoro-4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)thiocarbonyl!amino!-benzoate(Compound 62) in 1.0 ml EtOH and 1.0 ml of THF was added 55 mg NaOH (1.4mmol) and 1.0 ml of H₂ O. After stirring at room temperature forovernight EtOAc was added, and the reaction quenched by the addition of10% HCl. Extraction with EtOAc was followed by washing of the combinedorganic layers with H₂ O, saturated aqueous NaCl, and drying over MgSO₄.Removal of the solvent under reduced pressure provided a solid whichafter crystallization from CH₃ CN afforded the title compound as apale-yellow solid. 1H NMR (d₆ -acetone): δ 11.05 (1H, s), 8.02 (1H, m),7.99 (1H, t, J=8.3 Hz), 7.75 (1H, m), 7.69 (1H, dd, J=2.0, 6.1 Hz), 7.52(1H, s), 7.46 (1H, d, J=8.1 Hz), 7.21 (4H, m), 6.04 (1H, t, J=4.8 Hz),2.37 (2H, d, J=4.8 Hz), 2.33 (3H, s), 1.36 (6H, s).

Ethyl 5',6'-dihydro-5',5'-dimethyl-8'-(4-methylphenyl)-2,2'-binaphthalene!-6-carboxylate (Compound 64)

A solution of3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-7-bromonaphthalene CompoundD) 0.45 g, 1.40 mmol) and THF (2.1 ml) was added to magnesium turnings(0.044 g, 1.82 mmol) at room temperature under argon. Two drops ofethylene dibromide were added, and the solution, which slowly becamecloudy and yellow, was heated to reflux for 1.5 hours. In a second flaskwas added zinc chloride (0.210 g, 1.54 mmol), which was melted underhigh vacuum, cooled to room temperature and dissolved in THF (3 ml). TheGrignard reagent was added to the second flask and, after 30 minutes atroom temperature, a solution of ethyl 6-bromo-2-naphthalinecarboxylate(Compound N) 0.293 g, (1.05 mmol) and THF (2 ml) were added. In a thirdflask was prepared a solution of Ni(PPh₃)₄ and THF as follows: To asolution of NiCl₂ (PPh₃)₂ (0.82 g, 1.25 mmol) and PPh₃ (0.66 g, 2.5mmol) in THF (3.5 ml) was added a 1M solution of diisobutylaluminumhydride and hexanes (2.5 ml, 2.5 mmol), and the resulting solutiondiluted with THF to a total volume of 15 ml and stirred at roomtemperature for 15 minutes. Three 0.60 ml aliquots of the Ni(PPh₃)₄solution were added at 15 minutes intervals to the second flask. Theresulting suspension was stirred at room temperature for 2 hours. Thereaction was quenched by the addition of 5 ml 1N aqueous HCl and stirredfor 1 hour before extracting the products with ethyl acetate. Theorganic layers were combined, washed with brine, dried (MgSO₄), filteredand the solvent removed in-vacuo. The residue was crystalized fromhexanes to give 130 mg of pure material. The mother liquor wasconcentrated under reduced pressure and the residue purified by silicagel chromatography (95:5--hexanes:ethyl acetate) to give an additional170 mg of the title compound (overall yield=300 mg, 64%) as a colorlesssolid. 1H NMR (CDCl₃) δ 8.57 (s, 1H), 8.05 (dd, 1H, J=1.7, 8.0 Hz),7.84-7.95 (overlapping d's, 3H), 7.66 (dd, 1H, J=1.7, 8.5 Hz), 7.58 (dd,1H, J=2.0, 8.0 Hz), 7.48 (d, 1H, J=8.0 Hz), 7.43 (d, 1H, J=2.0 Hz), 7.32(d, 2H, J=8.0 Hz), 7.21 (d, 2H, J=8.0 Hz), 6.04 (t, 1H, J=4.8 Hz), 4.44(q, 2H, J=7.1 Hz), 2.40 (s, 3H), 2.39 (d, 2H, J=4.8 Hz), 1.45 (t, 3H,J=7.1 Hz), 1.39 (s, 6H).

5',6'-Dihydro-5',5'-dimethyl-8'-(4-methylphenyl)-2,2'-binaphthalenel-6-carboxylic acid (Compound 65)

A solution of ethyl 5',6'-dihydro-5',5'-dimethyl-8'-(4-methylphenyl)-2,2'-binaphthalene!-6-carboxylate (Compound 64) 0.19 g, 0.43 mmol), EtOH(8 ml) and 1N aqueous NaOH (2 ml) was heated to 60° C. for 3 hours. Thesolution was cooled to 0° C. and acidified with 1N aqueous HCl. Theproduct was extracted into ethyl acetate, and the organic layerscombined, washed with water, brine, dried (MgSO₄), filtered and thesolvent removed in-vacuo. The residue was recrystalized from THF/ethylacetate at 0° C. to give 35 mg of pure material. The mother liquor wasconcentrated under reduced pressure and the residue purified by silicagel chromatography (100% ethyl acetate) to give an additional 125 mg ofthe title compound (overall yield=160 mg, 90%) as a colorless solid. 1HNMR (DMSO-d₆) δ 8.57 (s, 1H), 8.11 (d, 1H, J=8.7 Hz), 7.96-7.82(overlapping d's, 3H),7.65 (d, 2H, J=7.6 Hz), 7.50 (d, 1H, J=7.9 Hz),7.28 (s, 1H), 7.26 (d, 2H, J=8.3 Hz), 7.21 (d, 2H, J=8.3 Hz), 6.01 (t,1H, J=4.5 Hz), 3.34 (br s, 1H), 2.31 (s, 3H), 2.31 (d, 2H, J=4.5 Hz),1.31 (s, 6H).

Ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-furyl)-2-naphthalenyl)ethynyl!benzoate(Compound 66)

Employing the same general procedure as for the preparation of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl)ethynyl!benzoate(Compound 1), 250.0 mg (0.52 mmol) of ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(trifluoromethylsulfonyl)oxy-2-naphthalenyl)ethynyl!benzoate(Compound G) was converted into the title compound (colorless solid)using 142.4 mg (1.045 mmol) of zinc chloride, 24.1 mg (0.02 mmol) oftetrakis(triphenylphosphine)palladium(0) and 2-lithiofuran (prepared bythe addition of 53.4 mg (0.52 ml, 0.78 mmol) of n-butyllithium (1.5Msolution in hexane) to a cold solution (-78° C.) of 53.4 mg (0.784 mmol)of furan in 1.0 ml of THF). PMR (CDCl₃): δ 1.32 (6H, s), 1.41 (3H, t,J=7.1 Hz), 2.35 (2H, d, J=5.0 Hz), 4.39 (2H, q, J=7.1 Hz), 6.41 (1H, t,J=5.0 Hz), 6.50 (2H, s), 7.36 (1H, d, J=8.0 Hz), 7.45 (1H, dd, J=1.7,8.0 Hz), 7.49 (1H, s), 7.57 (2H, d, J=8.2 Hz), 7.63 (1H, d, J=1.7 Hz),8.02 (2H, d, J=8.2 Hz).

4- (5,6-dihydro-5,5-dimethyl-8-(2-furyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 67)

Employing the same general procedure as for the preparation of 4-(5,6-dihydro-5,5-dimethyl-8-(2-thiazolyl)-2-naphthalenyl)ethynyl!benzoicacid (Compound 30a), ethyl 4-(5,6-dihydro-5,5-dimethyl-8-(2-furyl)-2-naphthalenyl)ethynyl!benzoate(Compound 66) was converted into the title compound (colorless solid)using 16.0 mg (0.38 mmol) of LiOH in H₂ O. PMR (d₆ -DMSO): δ 1.26 (6H,s), 2.33 (2H, d, J=4.9 Hz), 6.41 (1H, t, J=4.9 Hz), 6.60 (2H, m),7.45-7.53 (3H, m), 7.64 (2H, d, J=8.3 Hz), 7.75 (1H, d, J=1.6 Hz), 7.93(2H, d, J=8.3 Hz).

3,4-dihydro-4,4-dimethyl-7-acetyl-1(2H)-naphthalenone (Compound 100C)and 3,4-dihydro-4,4-dimethyl-6-acetyl-1(2H)-naphthalenone (Compound100D)

To a cold (0° C.) mixture of aluminum chloride (26.3 g, 199.0 mmols) indichloromethane (55 ml) was added acetylchloride (15 g, 192 mmols) and1,2,3,4-tetrahydro-1,1-dimethylnaphthalene (24.4 g, 152 mmols) indichloromethane (20 ml) over 20 minutes. The reaction mixture was warmedto ambient temperature and stirred for 4 hours. Ice (200 g) was added tothe reaction flask and the mixture diluted with ether (400 ml). Theaqueous and organic layers were separated and the organic phase waswashed with 10% HCl (50 ml), water (50 ml), 10% aqueous sodiumbicarbonate, and saturated aqueous NaCl (50 ml) and then dried overMgSO₄. The solvent was removed by distillation to afford a yellow oilwhich was dissolved in benzene (50 ml).

To a cold (0° C.) solution of acetic acid (240 ml) and acetic anhydride(120 ml) was added chromium trioxide (50 g, 503 mmols) in small portionsover 20 minutes under argon. The mixture was stirred for 30 minutes at0° C. and diluted with benzene (120 ml). The benzene solution preparedabove was added with stirring via an addition funnel over 20 minutes.After 8 hours, the reaction was quenched by the careful addition ofisopropanol (50 ml) at 0° C., followed by water (100 ml). After 15minutes, the reaction mixture was diluted with ether (1100 ml) and water(200 ml), and then neutralized with solid sodium bicarbonate (200 g).The ether layer was washed with water (100 ml),and saturated aqueousNaCl (2×100 ml), and dried over MgSO₄. Removal of the solvent underreduced pressure afforded a mixture of the isomeric diketones which wereseparated by chromatography (5% EtOAc/hexanes). (Compound 100C): 1H NMR(CDCl₃): δ 8.55 (1H, d, J=2.0 Hz), 8.13 (1H, dd, J=2.0, 8.3 Hz), 7.53(1H, d, J=8.3 Hz), 2.77 (2H, t, J=6.6 Hz), 2.62 (3H, s), 2.05 (2H, t,J=6.6 Hz), 1.41 (6H, s). (Compound 100D): 1H NMR (CDCl₃): δ 8.10 (1H, d,J=8.1 Hz), 8.02 (1H, d, J=1.6 Hz), 7.82 (1H, dd, J=1.6, 8.1 Hz), 2.77(2H, t, J=7.1 Hz), 2.64 (3H, s), 2.05 (2H, t, J=7.1 Hz), 1.44 (6H, s).

3,4-dihydro-4,4-dimethyl-6-(2-(2-methyl-13-dioxolanyl))-1(2H)-naphthalenone (Compound 100E)

A solution of 1.80 g (8.34 mmol) of a 1:5 mixture of3,4-dihydro-4,4-dimethyl-7-acetyl-1(2H)-naphthalenone (Compound 100C);and 3,4-dihydro-4,4-dimethyl-6-acetyl-1(2H)-naphthalenone (Compound100D) in 50 ml benzene was combined with 517.7 mg (8.34 mmol) ofethylene glycol and 20.0 mg (0.11 mmol) of p-toluenesulfonic acidmonohydrate. The resulting solution was heated to reflux for 18 hours,cooled to room temperature, and concentrated under reduced pressure. Thetitle compound was isolated by column chromatography (10%EtOAc--hexanes) as a colorless oil. 1H NMR (CDCl₃): δ 8.01 (1H, d, J=8.2Hz), 7.51 (1H, s), 7.43 (1H, dd, J=1.7, 6.4 Hz), 4.07 (2H, m), 3.79 (2H,m), 2.74 (2H, t, J=6.5 Hz), 2.04 (2H, t, J=7.1 Hz), 1.67 (3H, s), 1.46(6H, s).

1 2,34-tetrahydro-1-hydroxy-1-(4-methylphenyl)-4,4-dimethyl-6-(2-(2-methyl-1,3-dioxolanyl))naphthalene(Compound 100F)

To a solution of 496.2 mg (2.54 mmol) p-tolylmagnesiumbromide in 20 mlTHF (2.54 ml; 1M solution in ether) was added a solution of3,4-dihydro-4,4-dimethyl-6-(2-(2-methyl-1,3-dioxolan-yl))-1(2H)-naphthalenone(Compound 100E, 200.0 mg, 0.769 mmol) in THF (5 ml). The solution wasrefluxed for 16 hours, cooled to room temperature, and washed withwater, saturated aqueous NH₄ Cl, and dried over MgSO₄. Removal of thesolvents under reduced pressure and column chromatography (10%EtOAc/hexanes) afforded the title compound as a colorless solid. 1H NMR(CDCl₃): δ 7.49 (1H, d, J=1.7 Hz), 7.19 (2H, m), 7.10 (2H, d, J=7.9 Hz),7.04 (1H, d, J=8.2 Hz), 4.05 (2H, m), 3.80. (2H, m), 2.34 (3H, s), 2.21(1H, m), 2.10 (1H, m), 1.88 (1H, m), 1.65 (3H, s), 1.54 (1H, m), 1.39(3H, s), 1.33 (3H, s).

3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-6-acetylnaphthalene(Compound 100G)

A solution of1,2,3,4-tetrahydro-1-hydroxy-1-(4-methylphenyl)-4,4-dimethyl-6-(2-(2-methyl-1,3-dioxolanyl))naphthalene(Compound 100F 160.0 mg, 0.52 mmol), p-toluenesulfonic acid monohydrate(4 mg) and 30 ml benzene was refluxed for 12 hours. After cooling toroom temperature, the reaction mixture was diluted with ether (100 ml)and washed with 10% aqueous sodium bicarbonate, water, and saturatedaqueous NaCl. The organic layer was dried over MgSO₄ and the solventswere removed under reduced pressure to give the title compound, whichwas isolated by column chromatography (10% EtOAc-hexanes) as a yellowoil. 1H NMR (CDCl₃): δ 7.97 (1H, d, J=1.8 Hz), 7.67 (1H, dd, J=1.7, 6.4Hz), 7.22 (4H, s), 7.13 (1H, d, J=8.1 Hz), 6.10 (1H, t, J=4.5 Hz), 2.59(3H, s), 2.40 (3H, s), 2.38 (2H, d, J=4.7 Hz), 1.38 (6H, s).

4-3-oxo-3-(7,8-dihydro-5-(4-methylphenyl)-8,8-dimethyl-2-naphthalenyl)-1-propenyl!-benzoicacid (Compound 101)

To a solution of 78.7 mg (0.272 mmol)3,4-dihydro-1-(4-methylphenyl)-4,4-dimethyl-6-acetylnaphthalene(Compound 100G) in 4.0 ml of MeOH was added 53.1 mg (0.354 mmol) of4-carboxy benzaldehyde, and 80. mg (2.00 mmol; 2.0 ml of 1M aqueousNaOH). The resulting solution was stirred at room temperature for 12hours, concentrated under reduced pressure, and the residual oildissolved in EtOAc. The solution was treated with 10% HCl, and theorganic layer was washed with H₂ O, and saturated aqueous NaCl, thendried over Na₂ SO₄. Removal of the solvents under reduced pressure gavethe title compound as a colorless solid which was purified byrecrystallization from CH₃ CN. 1H NMR (acetone-d6): δ 8.00 (7H, m), 7.83(1H, d, J=15.6 Hz), 7.24 (4H, s), 7.13 (1H, d, J=8.1 Hz), 6.12 (1H, t,J=4.5 Hz), 2.42 (2H, d, J=4.8 Hz), 2.38 (3H, s), 1.41 (6H, s).

3,4-dihydro-1-phenyl-4,4-dimethyl-6-acetylnaphthalene (Compound 100H)

To a solution of 508.0 mg (1.95 mmol) of3,4-dihydro-4,4-dimethyl-6-(2-(2-methyl-1,3-dioxolanyl))-1(2H)-naphthalenone(Compound 100E) in 10 ml of THF was added 496.2 mg (2.54 mmol; 2.54 mlof a 1M solution in Et₂ O) of phenylmagnesium bromide. The resultingsolution was heated to reflux for 8 hours, H₂ O was added and heatingcontinued for 30 minutes. The THF was removed under reduced pressure andthe aqueous residue was extracted with EtOAc. The combined organiclayers were dried (MgSO₄), concentrated under reduced pressure, and thetitle compound isolated from the residue by column chromatography (10%EtOAc--hexanes) as a colorless oil. 1H NMR (CDCl₃): δ 7.97 (1H, d, J=1.8Hz), 7.67 (1H, dd, J=2.1, 8.0 Hz), 7.34 (5H, m), 7.10 (1H, d, J=8.1 Hz),6.12 (1H, d, J=4.6 Hz), 2.59 (3H, s), 2.39 (2H, d, J=4.8 Hz), 1.38 (6H,s).

4-3-oxo-3-(7,8-dihydro-5-phenyl-8,8-dimethyl-2-naphthalenyl)-1-propenyl!-benzoicacid (Compound 103)

To a solution of 115.0 mg (0.42 mmol) of3,4-dihydro-1-phenyl-4,4-dimethyl-6-acetylnaphthalene (Compound 100H)and 65.0 mg (0.43 mmol) of 4-formyl-benzoic acid in 5.0 ml EtOH and 1.0ml THF, was added 120.0 mg (3.00 mmol; 3.0 ml of a 1M aqueous solution)of NaOH. The resulting yellow solution was stirred at room temperaturefor 12 hours. The solution was acidified with 6% aqueous HCl andextracted with EtOAc. The combined organic layers were dried (MgSO₄),concentrated under reduced pressure, and the title compounds wasisolated by column chromatography (50% EtOAc--hexanes) as a pale yellowsolid. 1H NMR (CDCl₃): δ 8.13 (2H, d, J=7.7 Hz), 8.04 (1H, s), 7.81 (1H,d, J=15.5 Hz), 7.75 (3H, m), 7.60 (1H, d, J=15.5 Hz), 7.35 (5H, m), 7.14(1H, d, J=8.1 Hz), 6.15 (1H, t, J=4.2 Hz), 2.41 (2H, d, J=4.2 Hz), 1.41(6H, s).

Method of Potentiating Nuclear Receptor Agonists

Overview and introduction

We have discovered that a subset of retinoid antagonists which exhibitnegative hormone activity can be used for potentiating the biologicalactivities of other retinoids and steroid receptor superfamily hormones.These other retinoids and steroid receptor superfamily hormones can beeither endogenous hormones or pharmaceutical agents. Thus, for example,when used in combination with a retinoid negative hormone, certainactivities of pharmaceutical retinoid agonists can be rendered moreactive in eliciting specific biological effects. Advantageously, thiscombination approach to drug administration can minimize undesirableside effects of pharmaceutical retinoids because lower dosages of thepharmaceutical retinoids can be used with improved effectiveness.

More particularly, we have discovered that AGN 193109, a syntheticretinoid having the structure shown in FIG. 1, exhibits unique andunexpected pharmacologic activities. AGN 193109 exhibits high affinityfor the RAR subclass of nuclear receptors without activating thesereceptors or stimulating transcription of retinoid responsive genes.Instead, AGN 193109 inhibits the activation of RARs by retinoid agonistsand therefore behaves as a retinoid antagonist.

Additionally, we have discovered that retinoid negative hormones can beused without coadministration of a retinoid agonist or steroid hormoneto control certain disease symptoms. More specifically, the retinoidnegative hormone disclosed herein can down-regulate the high level basaltranscription of genes that are responsive to unliganded RARs. If, forexample, uncontrolled cellular proliferation results from the activityof genes responsive to unliganded RARs, then that gene activity can bereduced by the administration of a retinoid negative hormone thatinactivates RARs. Consequently, cellular proliferation dependent on theactivity of unliganded RARs can be inhibited by the negative hormone.Inhibition of unliganded RARs cannot be achieved using conventionalantagonists.

Significantly, we have discovered that AGN 193109 can both repress RARbasal activity and can sometimes potentiate the activities of otherretinoid and steroid receptor superfamily hormone agonists. In thecontext of the invention, a hormone agonist is said to be potentiated bya negative hormone such as AGN 193109 if, in the presence of thenegative hormone, a reduced concentration of the agonist elicitssubstantially the same quantitative response as that obtainable with theagonist alone. The quantitative response can, for example, be measuredin a reporter gene assay in vitro. Thus, a therapeutic retinoid thatelicits a desired response when used at a particular dosage orconcentration is potentiated by AGN 193109 if, in combination with AGN193109, a lower dosage or concentration of the therapeutic retinoid canbe used to produce substantially the same effect as a higher dosage orconcentration of the therapeutic retinoid when that therapeutic retinoidis used alone. The list of agonists that can be potentiated bycoadministration with AGN 193109 includes RAR agonists, vitamin Dreceptor agonists, glucocorticoid receptor agonists and thyroid hormonereceptor agonists. More particularly, specific agonists that can bepotentiated by coadministration include: ATRA, 13-cis retinoic acid, thesynthetic RAR agonist AGN 191183, 1,25-dihydroxyvitamin D₃,dexamethasone and thyroid hormone (3,3',5-triiodothyronine). Alsodisclosed herein is a method that can be used to identify other hormonesthat can be potentiated by coadministration with AGN 193109.

Thus, AGN 193109 behaves in a manner not anticipated for a simpleretinoid antagonist, but as a negative hormone that can potentiate theactivities of various members of the family of nuclear receptors. Wealso disclose a possible mechanism that can account for both negativehormone activity and the ability of AGN 193109 to potentiate theactivities of other nuclear receptor ligands. This mechanismincorporates elements known to participate in retinoid-dependentsignalling pathways and additionally incorporates a novel negativeregulatory component.

Those having ordinary skill in the art will appreciate that RARs, whichare high affinity targets of AGN 193109 binding, are transcriptionfactors that regulate the expression of a variety of retinoid responsivegenes. Cis-regulatory DNA binding sites for the RARs have beenidentified nearby genes that are transcriptionally regulated in aretinoid-dependent fashion. RAR binding to such DNA sites, known asretinoic acid response elements (RAREs), has been well defined.Importantly, the RAREs bind heterodimers consisting of one RAR and oneRXR. The RXR component of the heterodimer functions to promote a highaffinity interaction between the RAR/RXR heterodimer and the RARE(Mangelsdorf et al. The Retinoid Receptors in The Retinoids: Biology,Chemistry and Medicine, 2nd edition, eds. Sporn et al., Raven Press,Ltd., New York 1994, the disclosure of which is hereby incorporated byreference).

As detailed below, our findings related to the negative hormone activityof AGN 193109 are consistent with a mechanism involving the interactionof a putative Negative Coactivator Protein (NCP) with the RAR. Accordingto the proposed mechanism, this interaction is stabilized by AGN 193109.

Our results further indicated that AGN 193109 can modulate intracellularavailability of NCP for interaction with nuclear receptors other thanRARs that are occupied by AGN 193109. It follows that AGN 193109 canpotentiate transcriptional regulatory pathways involving nuclearreceptors that share with the RARs the ability to bind the NCP. In thisregard, AGN 193109 exhibits the ability to modulate a variety of nuclearreceptor pathways, an activity that would not be predicted for aconventional retinoid antagonist. Accordingly, AGN 193109 is useful asan agent for potentiating the activity of nuclear receptor ligands,including both endogenous hormones and prescribed therapeutics. Thisspecific embodiment illustrates the more general principle that anynuclear receptor negative hormone will potentiate the activity of othernuclear receptors that competitively bind the NCP.

Although other materials and methods similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described.General references for methods that can be used to perform the variousnucleic acid manipulations and procedures described herein can be foundin Molecular Cloning: A Laboratory Manual (Sambrook et al. eds. ColdSpring Harbor Lab Publ. 1989) and Current Protocols in Molecular Biology(Ausubel et al. eds., Greene Publishing Associates andWiley-Interscience 1987). The disclosures contained in these referencesare hereby incorporated by reference. A description of the experimentsand results that led to the creation of the present invention follows.

Example 6 describes the methods used to demonstrate that AGN 193109bound each of three RARs with high affinity but failed to activateretinoid dependent gene expression.

Example 6 AGN 193109 Binds RARs With High Affinity But Does NotTransactivate Retinoid-Dependent Gene Expression

Human RAR-α, RAR-β and RAR-γ receptors were separately expressed asrecombinant proteins using a baculovirus expression system essentiallyaccording to the method described by Allegretto et al. in J. Biol. Chem.268:26625 (1993). The recombinant receptor proteins were separatelyemployed for determining AGN 193109 binding affinities using the ³H!-ATRA displacement assay described by Heyman et al. in Cell 68:397(1992). Dissociation constants (Kds) were determined according to theprocedure described by Cheng et al. in Biochemical Pharmacology 22:3099(1973).

AGN 193109 was also tested for its ability to transactivate RARs in CV-1cells transiently cotransfected with RAR expression vectors and aretinoid responsive reporter gene construct. Receptor expression vectorspRShRAR-α (Giguere et al. Nature 330:624 (1987)), pRShRAR-β (Benbrook etal. Nature 333:669 (1988)) and pRShRAR-γ (Ishikawa et al. Mol.Endocrinol. 4:837 (1990)) were separately cotransfected with theΔMTV-TREp-Luc reporter plasmid. Use of this luciferase reporter plasmidhas been disclosed by Heyman et al. in Cell 68:397 (1992). TheΔMTV-TREp-Luc plasmid is essentially identical to the ΔMTV-TREp-CATreporter construct described by Umesono et al. in Nature 336:262 (1988),except that the chloramphenicol acetyltransferase (CAT) reporter genewas substituted by a polynucleotide sequence encoding fireflyluciferase. Transfection of green monkey CV-1 cells was carried outusing the calcium phosphate coprecipitation method described inMolecular Cloning: A Laboratory Manual (Sambrook et al. eds. Cold SpringHarbor Lab Publ. 1989). CV-1 cells were plated at a density of 4×10⁴/well in 12 well multiwell plates and transiently transfected with acalcium phosphate precipitate containing 0.7 μg of reporter plasmid and0.1 μg of receptor plasmid according to standard laboratory procedures.Cells were washed after 18 hours to remove the precipitate and refedwith Dulbecco's modified Eagle's medium (DMEM) (Gibco), containing 10%activated charcoal extracted fetal bovine serum (Gemini Bio-Products).Cells were treated with vehicle alone (ethanol) or AGN 193109 (10⁻⁹ to10⁻⁶ M) for 18 hours. Cell lysates were prepared in 0.1M KPO₄ (pH 7.8),1.0% TRITON X-100, 1.0 mM DTT, 2 mM EDTA. Luciferase activity wasmeasured as described by de Wet et al. in Mol. Cell. Biol. 7:725 (1987),using firefly luciferin (Analytical Luminescence Laboratory) and an EG&GBerthold 96-well plate luminometer. Reported luciferase valuesrepresented the mean±SEM of triplicate determinations.

The results presented in Table 11 indicated that AGN 193109 bound eachof RAR-α, RAR-β, and RAR-γ with high affinity, but did not activateretinoid-dependent gene expression. More specifically, AGN 193109 boundeach of the three receptors with Kd values in the 2-3 nM range. Despitethis tight binding, AGN 193109 failed to activate gene expression whencompared with inductions stimulated by ATRA. Accordingly, thehalf-maximal effective concentration of AGN 193109 (EC₅₀) wasunmeasurable. Although not presented in the Table, we also found thatAGN 193109 had no measurable affinity for the RXRs.

                  TABLE 11                                                        ______________________________________                                        AGN 193109 Binding and Transactivation of the RARs                                   RAR-α                                                                             RAR-β  RAR-γ                                      ______________________________________                                        EC.sub.50 (nM)                                                                         No Activity No Activity No Activity                                  K.sub.d (nM)                                                                           2           2           3                                            ______________________________________                                    

Example 7 describes the methods used to demonstrate that AGN 193109 isan antagonist of ATRA dependent gene expression.

Example 7 AGN 193109-Dependent Inhibition of RAR Transactivation by ATRA

The ability of AGN 193109 to antagonize ATRA mediated RAR activation wasinvestigated in CV-1 cells cotransfected by the calcium phosphatecoprecipitation method of Sambrook et al. (Molecular Cloning: ALaboratory Manual Cold Spring Harbor Lab Publ. 1989). Eukaryoticexpression vectors pRShRAR-α (Giguere et al. Nature 330:624 (1987)),pRShRAR-β (Benbrook et al. Nature 333:669 (1988)) and pRShRAR-γ(Ishikawa et al. Mol. Endocrinol. 4:837 (1990)) were cotransfected withthe Δ-MTV-Luc reporter plasmid described by Hollenberg et al. (Cell55:899 (1988)). Notably, the reporter plasmid contained two copies ofthe TRE-palindromic response element. Calcium phosphate transfectionswere carried out exactly as described in Example 6. Cells were dosedwith vehicle alone (ethanol), ATRA (10⁻⁹ to 10⁻⁶ M), AGN 193109 (10⁻⁹ to10⁻⁶ M), or 10⁻⁸ M ATRA in combination with AGN 193109 (10⁻⁹ to 10⁻⁶ M)for 18 hours. Cell lysates and luciferase activity measurements werealso performed as in Example 6.

The results of these procedures are presented in FIGS. 2A through 2Fwhere luciferase values represent the mean±SEM of triplicatedeterminations. More specifically, the results presented in FIGS. 2A, 2Cand 2E indicated that stimulation of transfected cells with ATRA led todose responsive increases in luciferase activity. This confirmed thatATRA activated each of the three RARs in the experimental system andprovided a comparative basis for detecting the activity of anantagonist. The graphic results presented in FIGS. 2B, 2D and 2Findicated that cotreatment of transfected cells with 10 nM ATRA andincreasing concentrations of AGN 193109 led to an inhibition ofluciferase activity. In particular, equal doses of AGN 193109 and ATRAgave greater than 50% inhibition relative to ATRA alone for all threeRAR subtypes. Comparison of the ATRA dose response in the presence ofdifferent concentrations of AGN 193109 indicated that ATRA wascompetitively inhibited by AGN 193109. Notably, the horizontal axes onall of the graphs shown in FIG. 2 represents the log of the retinoidconcentration. These results proved that AGN 193109 was a potent RARantagonist.

We next performed experiments to elucidate the mechanism underlying theantagonist activity of AGN 193109. Those having ordinary skill in theart will appreciate that nuclear receptor activation is believed toinvolve a conformational change of the receptor that is induced byligand binding. Indeed, the results of protease protection assays haveconfirmed that nuclear hormone agonists and antagonists cause receptorproteins to adopt different conformations (Keidel et al. Mol. Cell.Biol. 14:287 (1994); Allan et al. J. Biol. Chem. 267:19513 (1992)). Weused such an assay to determine if AGN 193109 and ATRA caused RAR-α toadopt different conformations. AGN 193583, an RAR-α-selectiveantagonist, was included as a positive control that is known to conferan antagonist-specific pattern of protease sensitivity.

Example 8 describes one method that was used to detect conformationalchanges in RAR-α resulting from AGN 193109 binding. As presented below,the results of this procedure unexpectedly indicated that AGN 193109 ledto a pattern of trypsin sensitivity that was substantially identical tothat induced by ATRA, an RAR agonist, and unlike that induced by a modelRAR antagonist. This finding suggested that AGN 193109 possessedproperties distinct from other retinoid antagonists.

Example 8 Protease Protection Analysis

A plasmid constructed in the vector pGEM3Z (Pharmacia) and containingthe RAR-α cDNA (Giguere et al. Nature 330:624 (1987)), was used inconnection with the TNT-coupled reticulocyte lysate in vitrotranscription-translation system (Promega) to prepare ³⁵ S!-methioninelabeled RAR-α. Limited proteolytic digestion of the labeled proteinRAR-α was carried out according to the method described by Keidel et al.in Mol. Cell. Biol. 14:287 (1994). Aliquots of reticulocyte lysatecontaining ³⁵ S!-methionine labeled RAR-α were incubated with eitherATRA, AGN 193583 or AGN 193109 on ice for 45 minutes in a total volumeof 9 μl. The retinoid final concentration for all trials was 100 nM forATRA and AGN 193109, and 1000 nM for AGN 193583. The difference betweenthe final concentrations of the retinoids was based on the approximate10-fold difference in relative affinities of ATRA and AGN 193109 (havingKd at RAR-α of 2 and 10 nM, respectively) and AGN 193583 (having Kd atRAR-α of ≧100 nM). After ligand binding, 1 μl of appropriatelyconcentrated trypsin was added to the mixture to give finalconcentrations of 25, 50 or 100 μg/ml. Samples were incubated at roomtemperature for 10 minutes and trypsin digestion stopped by addition ofSDS-sample buffer. Samples were subjected to polyacrylamide gelelectrophoresis and autoradiographed according to standard procedures.

Both the agonist and antagonist led to distinct patterns of trypsinsensitivity that were different from the result obtained by digestion ofthe unliganded receptor. Autoradiographic results indicated that trypsinconcentrations of 25, 50 and 100 μg/ml completely digested theradiolabeled RAR-α in 10 minutes at room temperature in the absence ofadded retinoid. Prebinding of ATRA led to the appearance of two majorprotease resistant species. Prebinding of the RAR-α-selective antagonistAGN 193583 gave rise to a protease resistant species that was of lowermolecular weight than that resulting from ATRA prebinding. This resultdemonstrated that a retinoid agonist and antagonist led toconformational changes detectable by virtue of altered trypsinsensitivities. Surprisingly, prebinding of AGN 193109 gave rise to aprotease protection pattern that was indistinguishable from thatproduced by prebinding of ATRA.

The results presented above confirmed that AGN 193109 bound the RAR-αand altered its conformation. Interestingly, the nature of thisconformational change more closely resembled that which resulted frombinding of an agonist (ATRA) than the alteration produced by antagonist(AGN 193583) binding. Clearly, the mechanism of AGN 193109 dependentantagonism was unique.

We considered possible mechanisms that could account for the antagonistactivity of AGN 193109. In particular, we used a standard gel shiftassay to test whether AGN 193109 perturbed RAR/RXR heterodimer formationor inhibited the interaction between RAR and its cognate DNA bindingsite.

Example 9 describes a gel electrophoretic mobility-shift assay used todemonstrate that AGN 193109 neither inhibited RAR/RXR dimerization norinhibited binding of dimers to a target DNA.

Example 9 Gel Shift Analysis

In vitro translated RAR-α was produced essentially as described underExample 8, except that ³⁵ S-labeled methoinine was omitted. In vitrotranslated RXR-α was similarly produced using apBluescript(II)(SK)-based vector containing the RXR-α cDNA described byMangelsdorf, et al. in Nature 345:224-229 (1990) as the template forgenerating in vitro transcripts. The labeled RAR-α and RXR-α, alone orin combination, or prebound with AGN 193109 (10⁻⁶ M) either alone or incombination, were allowed to interact with an end-labeled DR-5 RAREdouble-stranded probe having the sequence 5'-TCAGGTCACCAGGAGGTCAGA-3'(SEQ ID NO:1). The binding mixture was electrophoresed on anon-denaturing polyacrylamide gel and autoradiographed according tostandard laboratory procedures. A single retarded species appearing onthe autoradiograph that was common to all the lanes on the gelrepresented an undefined probe-binding factor present in thereticulocyte lysate. Only the RAR/RXR combination gave rise to aretinoid receptor-specific retarded species. Neither RAR alone nor RXRalone bound the probe to produce this shifted species. The presence ofAGN 193109 did not diminish this interaction.

These results indicated that AGN 193109 did not substantially altereither the homo- or hetero-dimerization properties of RAR-α. Further,AGN 193109 did not inhibit the interaction of receptor dimers with a DNAsegment containing the cognate binding site.

In view of the unique properties which characterized AGN 193109, weproceeded to investigate whether this antagonist could additionallyinhibit the activity of unliganded RARs. The receptor/reporter systemused to make this determination advantageously exhibited high levelconstitutive activity in the absence of added retinoid agonist. Morespecifically, these procedures employed the ER-RAR chimeric receptor andERE-tk-Luc reporter system. The ERE-tk-Luc plasmid includes the region-397 to -87 of the estrogen responsive 5'-flanking region of the Xenopusvitellogenin A2 gene, described by Klein-Hitpass, et al. in Cell46:1053-1061 (1986), ligated upstream of the HSV thymidine kinasepromoter and luciferase reporter gene of plasmid tk-Luc. The ER-RARchimeric receptors consisted of the estrogen receptor DNA binding domainfused to the "D-E-F" domain of the RARs. Those having ordinary skill inthe art appreciate this "D-E-F" domain functions to bind retinoid, toprovide a retinoid inducible transactivation function and to provide acontact site for heterodimerization with RXR. Thus, luciferaseexpression in this reporter system was dependent on activation of thetransfected chimeric receptor construct.

Example 10 describes the method used to demonstrate that AGN 193109inhibited basal gene activity attributable to unliganded RARs. Theseprocedures were performed in the absence of added retinoid agonist. Theresults presented below provided the first indication that AGN 193109exhibited negative hormone activity.

Example 10 Repression of Basal Gene Activity of a Retinoid-RegulatedReporter in Transiently Cotransfected Cell Lines

CV-1 cells were co-transfected with the ERE-tk-Luc reporter plasmid andeither ER-RAR-α, ER-RAR-β or ER-RAR-γ expression plasmids. TheERE-tk-Luc plasmid contained the estrogen-responsive promoter element ofthe Xenopus laevis vitellogenin A2 gene and was substantially identicalto the reporter plasmid described by Klein-Hitpass et al. in Cell46:1053 (1986), except that the CAT reporter gene was substituted by apolynucleotide sequence encoding luciferase. The ER-RAR-α, ER-RAR-β andER-RAR-γ chimeric receptor-encoding polynucleotides employed in theco-transfection have been described by Graupner et al. in Biochem.Biophys. Res. Comm. 179:1554 (1991). These polynucleotides were ligatedinto the pECE expression vector described by Ellis et al. in Cell 45:721(1986) and expressed under transcriptional control of the SV-40promoter. Calcium phosphate transfections were carried out exactly asdescribed in Example 6 using 0.5 μ/well of reporter plasmid and either0.05 μg, 0.10 μg or 0.2 μg/well of receptor plasmid. Cells were dosedwith vehicle alone (ethanol), ATRA (10⁻⁹ to 10⁻⁶ M), or AGN 193109 (10⁻⁹to 10⁻⁶ M) for 18 hours. Cell lysates and luciferase activitymeasurements were performed as described in Example 6.

The results presented in FIGS. 3A, 4A and 5A confirmed that ATRAstrongly induced luciferase expression in all transfectants. Basal levelexpression of luciferase for the three transfected chimeric RAR isoformsranged from approximately 7,000 to 40,000 relative light units (rlu) andwas somewhat dependent on the amount of receptor plasmid used in thetransfection. Thus, the three chimeric receptors were activatable byATRA, as expected. More specifically, all three receptors bound ATRA andactivated transcription of the luciferase reporter gene harbored on theERE-tk-Luc plasmid.

FIGS. 3B, 4B and 5B present AGN 193109 dose response curves obtained inthe absence of any exogenous retinoid agonist. Interestingly, ER-RAR-α(FIG. 3B) was substantially unaffected by AGN 193109, while the ER-RAR-βand ER-RAR-γ chimeric receptors (FIGS. 4B and 5B, respectively)exhibited an AGN 193109 dose responsive decrease in luciferase reporteractivity.

We further investigated the negative hormone activity of AGN 193109 bytesting its ability to repress gene expression mediated by a chimericRAR-γ receptor engineered to possess a constitutive transcriptionactivator domain. More specifically, we used a constitutively activeRAR-γ chimeric receptor fused to the acidic activator domain of HSVVP-16, called RAR-γ-VP-16, in two types of luciferase reporter systems.The first consisted of the ERE-tk-Luc reporter cotransfected withER-RARs and ER-RXR-α. The second utilized the AMTV-TREp-Luc reporterinstead of the ERE-tk-Luc reporter.

Example 11 describes the method used to demonstrate that AGN 193109could suppress the activity of a transcription activator domain of anRAR. The results presented below proved that AGN 193109 could suppressRAR-dependent gene expression in the absence of an agonist and confirmedthat AGN 193109 exhibited negative hormone activity.

Example 11 Repression of RAR-VP-16 Activity in Transiently TransfectedCells

CV-1 cells were transiently cotransfected according to the calciumphosphate coprecipitation technique described under Example 6 using 0.5μg/well of the ERE-tk-Luc luciferase reporter plasmid, 0.1 μg/well ofthe ER-RXR-α chimeric reporter expression plasmid, and either 0 μg or0.1 μg/well of the RAR-γ-VP-16 expression plasmid. The chimeric receptorER-RXR-α consisted of the hormone binding domain (amino acids 181 to458) of RXR-α (Mangelsdorf, et al. Nature 345:224-229 (1990)) fused tothe estrogen receptor DNA binding domain (Graupner, et al. Biochem.Biophys. Res. Comm. 179:1554 (1991)) and was expressed from the SV-40based expression vector pECE described by Ellis, et al. in Cell 45:721(1986). RAR-γ-VP-16 is identical to the VP16RAR-γ1 expression plasmiddescribed by Nagpal et al. in EMBO J. 12:2349 (1993), the disclosure ofwhich is hereby incorporated by reference, and encodes a chimericprotein having the activation domain of the VP-16 protein of HSV fusedto the amino-terminus of full length RAR-γ. At eighteen hourspost-transfection, cells were rinsed with phosphate buffered saline(PBS) and fed with DMEM (Gibco-BRL) containing 10% FBS (GeminiBio-Products) that had been extracted with charcoal to remove retinoids.Cells were dosed with an appropriate dilution of AGN 193109 or ATRA inethanol vehicle or ethanol alone for 18 hours, then rinsed with PBS andlysed using 0.1M KPO₄ (pH 7.8), 1.0% TRITON X-100, 1.0 mM DTT, 2 mMEDTA. Luciferase activity was measured according to the method describedby de Wet, et al. in Mol. Cell. Biol. 7:725 (1987), using fireflyluciferin (Analytical Luminescence Laboratory) and an EG&G Berthold96-well plate luminometer. Luciferase values represented the mean±SEM oftriplicate determinations.

As shown in FIG. 6, CV-1 cells transfected with the ERE-tk-Luc reporterconstruct and the ER-RAR-α chimeric expression plasmid exhibited a weakactivation of luciferase activity by ATRA, likely due to isomerizationof ATRA to 9C-RA, the natural ligand for the RXRs (Heyman et al. Cell68:397 (1992). Cells transfected with the same mixture of reporter andchimeric receptor plasmids but treated with AGN 193109 did not exhibitany effect on luciferase activity. As AGN 193109 does not bind to theRXRs, this latter result was expected. CV-1 cells similarly transfectedwith the ERE-tk-Luc reporter but with substitution of an ER-RAR chimericreceptor expression plasmid for ER-RXR-α exhibited a robust induction ofluciferase activity following ATRA treatment.

In contrast, inclusion of the RAR-γ-VP-16 expression plasmid with theER-RXR-α and ERE-tk-Luc plasmids in the transfection mixture resulted ina significant increase in the basal luciferase activity as measured inthe absence of any added retinoid. This increase in basal luciferaseactivity observed for the ER-RXR-α/RAR-γ-VP-16 cotransfectants, whencompared to the result obtained using cells transfected with ER-RXR-αalone, indicated that recombinant ER-RXR-α and RAR-γ-VP-16 proteinscould heterodimerize. Interaction of the heterodimer with thecis-regulatory estrogen responsive element led to a targeting of theVP-16 activation domain to the promoter region of the ERE-tk-Lucreporter. Treatment of such triply transfected cells with ATRA led to amodest increase of luciferase activity over the high basal level.However, treatment of the triple transfectants with AGN 193109 resultedin a dose dependent decrease in luciferase activity. Importantly, FIG. 6shows that AGN 193109 treatment of cells cotransfected with ER-RXR-α andRAR-γ-VP-16 led to repression of luciferase activity with maximalinhibition occurring at approximately 10⁻⁸ M AGN 193109.

Our observation that AGN 193109 repressed the constitutivetranscriptional activation function of RAR-γ-VP-16 in the presence of anRXR was explained by a model wherein binding of AGN 193109 to the RARinduced a conformational change in the RAR which stabilizes a negativeconformation that facilitates the binding of a trans-acting negativecoactivator protein. When the AGN 193109/RAR complex is bound by theNCP, the RAR is incapable of upregulating transcription of genes thatare ordinarily responsive to activated RARs. Our model further proposesthat the intracellular reservoir of NCP is in limiting concentration incertain contexts and can be depleted by virtue of AGN 193109 stimulatedcomplexation with RARs.

The results presented in FIG. 6 additionally indicated that even at 10⁻⁶M AGN 193109, the ER-RXR-α and RAR-γ-VP-16 proteins could interact toform heterodimers competent for activating transcription of the reportergene. More specifically, cells transfected with ER-RXR-α and RAR-γ-VP-16and treated with AGN 193109 at a concentration (10⁻⁸ -10⁻⁶ M) sufficientto provide maximal inhibition, gave luciferase activity readings ofapproximately 16,000 rlu. Conversely, cells transfected only withER-RXR-α and then treated with AGN 193109 at a concentration as high as10⁻⁶ M exhibited luciferase expression levels of only approximately8,000 rlu. The fact that a higher level of luciferase activity wasobtained in cells that expressed both ER-RXR-α and RAR-γ-VP-16, even inthe presence of 10⁻⁶ M AGN 193109 demonstrated the persistence of aninteraction between the two recombinant receptors. The repression ofRAR-γ-VP-16 activity by AGN 193109 suggested that modulation of NCPinteraction can be codominate with VP-16 activation. Accordingly, werealized that it may be possible to modulate the expression of geneswhich are not ordinarily regulated by retinoids in an AGN 193109dependent manner.

Candidates for AGN 193109 regulatable genes include those that areactivated by transcription factor complexes which consist of non-RARfactors that associate or heterodimerize with RARs, wherein the non-RARfactor does not require an RAR agonist for activation. While stimulationwith an RAR agonist may have substantially no effect on the expressionof such genes, administration with AGN 193109 can promote formation ofinactive transcription complexes comprising AGN 193109/RAR/NCP.Consequently, addition of the AGN 193109 retinoid negative hormone candown-regulate transcription of an otherwise retinoid-insensitive gene.

This same mechanism can account for the observation that AGN 193109 canrepress the activity of the tissue transglutaminase (TGase) gene inHL-60 cells. A retinoid response element consisting of three canonicalretinoid half sites spaced by 5 and 7 base pairs has been identified inthe transcription control region of this gene. While TGase can beinduced by RXR-selective agonists, it is not responsive to RAR-selectiveagonists. The TGase retinoid response element is bound by an RAR/RXRheterodimer (Davies et al. in Press). Interestingly, AGN 193109 is ableto repress TGase activity induced by RXR agonists. This AGN 193109mediated repression can be accounted for by the ability of this negativehormone to sequester NCPs to the RAR component of the heterodimer,thereby repressing the activity of the associated RXR.

We have also obtained results which support conclusions identical tothose presented under Example 11 by employing RAR-γ-VP-16 and expressionconstructs and the ΔMTV-TREp-Luc reporter plasmid instead of theRAR-γ-VP-16 and ER-RXR-α expression constructs in combination with theERE-tk-Luc reporter plasmid. Consistent with the results presentedabove, we found that RAR-γ-VP-16 activity at the ΔMTV-TREp-Luc reporterwas inhibited by AGN 193109. Therefore, AGN 193109 repressed RAR-γ-VP-16activity when this chimeric receptor was directly bound to a retinoicacid receptor response element instead of indirectly bound to anestrogen response element in the promoter region of the reporterplasmid. These findings demonstrated that an assay for identifyingagents having negative hormone activity need not be limited by the useof a particular reporter plasmid. Instead, the critical feature embodiedby an experimental system useful for identifying retinoid negativehormones involves detecting the ability of a compound to repress theactivity of an RAR engineered to contain a constitutive transcriptionactivation domain.

Generally, retinoid negative hormones can be identified as the subset ofretinoid compounds that repress within a transfected cell the basallevel expression of a reporter gene that is transcriptionally responsiveto direct or indirect binding by a retinoid receptor or a chimericreceptor that includes at least the domains of the retinoid receptorlocated C-terminal to the DNA binding domain of that receptor. Thisapproach has been adapted to a screening method useful for identifyingretinoid negative hormones. In the various embodiments of the inventedscreening method, the structure of the receptor for which a negativehormone is sought is variable. More specifically, the retinoid receptorcan be either of the RAR or the RXR subtype. The receptor can optionallybe engineered to include a constitutive transcription activator domain.The retinoid receptor used to screen for negative hormones optionallycontains a heterologous DNA binding domain as a substitute for the DNAbinding domain endogenous to the native receptor. However, when a secondreceptor is used in the screening method, and where the second receptorcan dimerize with the retinoid receptor for which a negative hormone issought, then that retinoid receptor may not require a DNA binding domainbecause it can be linked to the transcription control region of thereporter gene indirectly through dimerization with the second receptorwhich is itself bound to the transcription control region.

In the practice of the screening method, the ability of a compound torepress the basal expression of a reporter is typically measured in anin vitro assay. Basal expression represents the baseline level ofreporter expression in transfected cells under conditions where noexogenously added retinoid agonist is present. Optionally, steps may betaken to remove endogenous retinoid ligands from the environment of thetransfected cells via procedures such as charcoal extraction of theserum that is used to culture cells in vitro.

Examples of reporter genes useful in connection with the screeningmethod include those encoding luciferase, beta galactosidase,chloramphenicol acetyl transferase or cell surface antigens that can bedetected by immunochemical means. In practice, the nature of thereporter gene is not expected to be critical for the operability of themethod. However, the transcriptional regulatory region of the reporterconstruct must include one or more cis-regulatory elements that aretargets of transcription factors for which negative hormones are beingsought. For example, if one desires to identify RAR negative hormones,then the transcriptional regulatory region of the reporter constructcould contain a cis-regulatory element that can be bound by anRAR-containing protein. In this example, there should be correspondencebetween the DNA binding domain of the RAR and the cis-regulatory elementof the transcriptional regulatory region of the reporter construct.Thus, if a chimeric RAR having a constitutive transcription activatordomain and a DNA binding domain that can bind cis-regulatory estrogenresponsive elements is employed in the screening method, then thetranscriptional regulatory region of the reporter construct shouldcontain an estrogen responsive element.

Examples of cis-regulatory elements that directly bind retinoidreceptors (RAREs) useful in connection with the reporter assay aredisclosed by Mangelsdorf et al. in The Retinoid Receptors in TheRetinoids: Biology, Chemistry and Medicine, 2nd edition, eds. Sporn etal., Raven Press, Ltd., New York (1994), the disclosure of which hasbeen incorporated by reference hereinabove. Examples of cis-regulatoryelements that indirectly bind chimeric receptors include DNA bindingsites for any DNA binding protein for which the DNA binding domain ofthe protein can be incorporated into a chimeric receptor consisting ofthis DNA binding domain attached to a retinoid receptor. Specificexamples of heterologous DNA binding domains that can be engineered intochimeric receptors and that will recognize heterologous cis-regulatoryelements include those recognizing estrogen responsive elements. Thus,the retinoid receptor portion of a chimeric receptor useful inconnection with the screening method need not contain the DNA binding ofthe retinoid receptor but must contain at least the ligand bindingdomain of the retinoid receptor.

A further example of indirect retinoid receptor binding to thecis-regulatory element includes the use of a protein that can bind thecis-regulatory element and dimerize with a retinoid receptor. In thiscase, the retinoid receptor associates with the cis-regulatory elementonly by association with the protein responsible for DNA binding. Anexample of such a system would include the use of a fusion proteinconsisting of a heterologous DNA binding domain fused to an RXR,containing at least the domain of the RXR responsible for dimerizationwith RARS. Cointroduced RARs can dimerize with such a fusion proteinbound to the cis-regulatory element. We anticipate that anycis-regulatory element-binding protein that dimerizes with RARs toresult in an indirect association of the RAR with the cis-regulatoryelement will also be suitable for use with the negative hormonescreening method.

In a preferred embodiment of the screening method, retinoid negativehormones are identified as those retinoids that repress basal expressionof an engineered RAR transcription factor having increased basalactivity. Although not essential for operability of the screeningmethod, the engineered RAR employed in the following Example included aconstitutive transcription activating domain. Use of this chimericreceptor advantageously provided a means by which the basal expressionof a reporter gene could be elevated in the absence of any retinoid.Although we have employed transient transfection in the proceduresdetailed above, stably transfected cell lines constitutively expressingthe chimeric receptor would also be useful in connection with thescreening method.

As disclosed in the following Example, a chimeric retinoid receptorhaving a constitutive transcription activator domain washeterodimerizable with a second receptor engineered to contain a DNAbinding domain specific for an estrogen responsive cis-regulatoryelement. In this case the chimeric retinoid receptor having aconstitutive transcription activator domain associates with thecis-regulatory region controlling reporter gene expression indirectlyvia binding to a second receptor that binds a DNA target sequence. Moreparticularly, the second receptor was engineered to contain a DNAbinding domain that recognized an estrogen responsive element.Advantageously, the reporter gene having an estrogen responsive elementin the upstream promoter region was unresponsive to retinoid agonists inthe absence of the transfected chimeric receptor having the constitutivetranscription activator domain. Accordingly, all reporter gene activitywas attributed to the transfected receptors. The combination use of theestrogen responsive element DNA binding domain and the estrogenresponsive element cis-regulatory element are intended to beillustrative only. Those having ordinary skill in the art will realizethat other combinations of engineered receptors having specificity fornon-RARE cis-regulatory elements will also be useful in the practice ofthe invented screening method.

Cells useful in connection with the screening method will be eukaryoticcells that can be transfected. The cells may be animal cells such ashuman, primate or rodent cells. We have achieved very good results usingCV-1 cells, but reasonably expect that other cultured cell lines couldalso be used successfully. Any of a number of conventional transfectionmethods known in the art can be used to introduce an expressionconstruct encoding the chimeric retinoid receptor having a constitutivetranscription activator domain.

The constitutive transcription activator domain will consist of aplurality of amino acids which will likely have an overall acidiccharacter as represented by a negative charge under neutral pHconditions. For example, the constitutive transcription activator domainmay have an amino acid sequence which is also found in viraltranscription factors. One example of a viral transcription factorhaving a constitutive transcription activator domain is the herpessimplex virus 16. However, other viral or synthetic transcriptionactivator domains would also be useful in the construction of expressionconstructs encoding the chimeric retinoid receptor having a constitutivetranscription activator domain.

As described below, we have developed a generalized screening methoduseful for identifying retinoid negative hormones. This screening methodprovides a means for distinguishing simple antagonists from negativehormones. Table 12 lists several retinoid compounds which exhibit potentaffinity for RAR-γ yet, with the exception of ATRA, did nottransactivate this receptor in a transient cotransfectiontransactivation assay. We therefore tested these compounds to determinewhich were RAR-γ antagonists and which, if any, of these antagonistsexhibited negative hormone activity.

Example 12 describes the method used to identify retinoid compounds thatwere antagonists, and the subset of antagonists that exhibited negativehormone activity.

Example 12 Assay for Retinoid Negative Hormones

4×10⁴ CV-1 cells were transfected by the calcium phosphatecoprecipitation procedure described in Molecular Cloning: A LaboratoryManual (Sambrook et al. eds. Cold Spring Harbor Lab Publ. 1989) using0.5 μg ERE-tk-Luc reporter plasmid and 0.1 μg ER-RAR-γ (Graupner et al.Biochem. Biophys. Res. Comm. 179:1554 (1991)) chimeric expressionplasmid. After 18 hours, cells were rinsed with PBS and fed with DMEM(Gibco-BRL) containing 10% activated charcoal extracted FBS (GeminiBio-Products). Cells were treated with 10⁻⁸ M ATRA in ethanol or ethanolalone. In addition, ATRA treated cells were treated with 10⁻⁹, 10⁻⁸,10⁻⁷ or 10⁻⁶ M of the compounds listed in Table 12. After 18 hours,cells were rinsed in PBS and lysed in 0.1M KPO₄ (pH 7.8), 1.0% TRITONX-100, 1.0 mM DTT, 2 mM EDTA. Luciferase activities were measured asdescribed by deWet et al. in Mol Cell. Biol. 7:725 (1987).

                  TABLE 12                                                        ______________________________________                                        Compound    Kd (nM) @ RAR-γ.sup.a                                                                EC.sub.50 (nM) @ RAR-γ.sup.a                   ATRA        12           17                                                   AGN 193109  6            na                                                   (Compound 60)                                                                 AGN 193174  52           na                                                   (Compound 34a)                                                                AGN 193199  30           na                                                   AGN 193385  25           na                                                   (Compound 23)                                                                 AGN 193389  13           na                                                   (Compound 25)                                                                 AGN 193840  40           na                                                   AGN 193871  30           na                                                   (Compound 50)                                                                 ______________________________________                                         .sup.a Relative affinity (Kd) determined by competition of .sup.3 HATRA       binding to baculovirus expressed RARγ and application of the            ChengPrussof equation.                                                        .sup.b EC.sub.50 measured in CV1 cells transiently cotransfected with         ΔMTVTREp-Luc and RSRAR-γ.                                         "na" denotes no activity.                                                

As indicated by the results presented in part in FIG. 7 and in Table 12,with the exception of ATRA, all of the compounds listed in Table 12 wereretinoid antagonists at RAR-γ.

The RAR-γ antagonists identified in Table 12 were next screened todetermine which, if any, were also retinoid negative hormones. 4×10⁴CV-1 cells were transfected according to the calcium phosphate proceduredescribed in Molecular Cloning: A Laboratory Manual (Sambrook et al.eds. Cold Spring Harbor Lab Publ. 1989) using 0.5 μg ERE-tk-Luc reporterplasmid and 0.1 μg ER-RXR-α (Graupner et al. Biochem. Biophys. Res.Comm. 179:1554 (1991)) and 0.2 μg RAR-γ-VP-16 (Nagpal et al. EMBO J.12:2349 (1993)) chimeric expression plasmids. After 18 hours, cells wererinsed with PBS and fed with DMEM (Gibco-BRL) containing 10% activatedcharcoal extracted FBS (Gemini Bio-Products). Cells were treated with10⁻⁹, 10⁻⁸, 10⁻⁷ or 10⁻⁶ M of each of the compounds listed in Table 12.Treatment with ethanol vehicle alone served as the negative control.After 18 hours, cells were rinsed in PBS and lysed in 0.1M KPO₄ (pH7.8), 1.0% TRITON X-100, 1.0 mM DTT, 2 mM EDTA. Luciferase activitieswere measured as previously by deWet et al. in Mol Cell. Biol. 7:725(1987).

As shown in FIG. 8, the retinoid antagonists of Table 12 could beseparated into two classes by virtue of their effect on the constitutivetranscription activation function of the RAR-γ-VP-16 chimeric retinoidreceptor. One group, which included AGN 193174, AGN 193199 and AGN193840, did not repress RAR-γ-VP-16 activity even though they were ATRAantagonists. In contrast AGN 193109, AGN 193385, AGN 193389 and AGN193871 exhibited a dose dependent repression of RAR-γ-VP-16 constitutiveactivity. Therefore, while the compounds of both groups were RAR-γantagonists, only those of the second group exhibited negative hormoneactivity. This assay advantageously distinguished retinoid negativehormones from simple retinoid antagonists.

The foregoing experimental results proved that AGN 193109 met thecriteria that define a negative hormone. More specifically, the resultspresented under Example 11 demonstrated that AGN 193109 had the capacityto exert inhibitory activity at the RARs even in the absence ofexogenously added retinoid ligands. As such, this compound possessedbiological activities that did not depend upon blockade of theinteraction between the RARs and agonists such as ATRA and AGN 191183.These findings led us to conclude that AGN 193109 stabilizedinteractions between RARs and NCPs. As diagrammed in FIG. 9, NCP/RAR/PCPinteractions exist in an equilibrium state. An agonist serves toincrease PCP interactions and decrease NCP interactions, while aninverse agonist or negative hormone stabilizes NCP and decreases PCPinteractions. As indicated previously, our experimental resultssuggested that the intracellular availability of NCP for other receptorscan be modulated by AGN 193109 administration. More specifically, wediscovered that AGN 193109 can promote complexation of NCP with RARs,thereby reducing the intracellular reservoir of NCP available forinteraction with transcription factors other than the RARs.

We next examined the effect of AGN 193109 on agonist-mediated inhibitionof AP-1 dependent gene expression. In Endocr. Rev. 14:651 (1993), Pfhaldisclosed that retinoid agonists can down-regulate gene expression by amechanism that involved inhibition of AP-1 activity. We postulated thatAGN 193109 could have had either of two effects when used in combinationwith a retinoid agonist in a model system designed to measure AP-1activity. First, AGN 193109 could conceivably have antagonized theeffect of the agonist, thereby relieving the agonist-dependentinhibition of AP-1 activity. Alternatively, AGN 193109 could havepotentiated the agonist's activity, thereby exaggerating theagonist-dependent inhibition of AP-1 activity.

Example 13 describes the methods used to demonstrate that AGN 193109potentiated the anti-AP-1 activity of a retinoid agonist. As disclosedbelow, the AGN 191183 retinoid agonist weakly inhibited AP-1 dependentgene expression. The combination of AGN 193109 and the retinoid agoniststrongly inhibited AP-1 dependent gene expression. By itself, AGN 193109had substantially no anti-AP-1 activity.

Example 13 AGN 193109 Potentiates the Anti-AP-1 Activity of a RetinoidAgonist

HeLa cells were transfected with 1 μg of the Str-AP1-CAT reporter geneconstruct and 0.2 μg of plasmid pRS-hRARα, described by Giguere et al.in Nature 33:624 (1987), using LIPOFECTAMINE (Life Technologies, Inc.).Str-AP1-CAT was prepared by cloning a DNA fragment corresponding topositions -84 to +1 of the rat stromelysin-1 promoter (Matrisian et al.,Mol. Cell. Biol. 6:1679 (1986)) between the HindIII-BamHI sites ofpBLCAT3 (Luckow et al., Nucl. Acids Res. 15:5490 (1987)). This sequenceof the stromelysin-1 promoter contains an AP1 motif as its sole enhancerelement (Nicholson et al., EMBO J. 9:4443 (1990). The promoter sequencewas prepared by annealing two synthetic oligonucleotides havingsequences: 5'-AGAAGCTTATGGAAGCAATTATGAGTCAGTTTGCGGGTGACTCTGCAAATACTGCCACT CTATAAAAGTTGGGCTCAGAAAGGTGGACCTCGAGGATCCAG-3'

(SEQ ID NO:2), and 5'-CTGGATCCTCGAGGTCCACCTTTCTGAGCCCAACTTTTATAGAGTGGCAGTATTTGCAGAGTCACCCGCAAACTGACTCATAATTGCTTCCATAAGCTTCT-3' (SEQ ID NO:3).Procedures involving transfection, treatment with appropriate compoundsand measurement of CAT activity were carried out as described by Nagpalet al. in J. Biol. Chem. 270:923 (1995), the disclosure of which ishereby incorporated by reference.

The results of these procedures indicated that AGN 193109 potentiatedthe anti-AP-1 activity of the retinoid agonist, AGN 191183. Morespecifically, in the concentration range of from 10⁻¹² to 10⁻¹⁰ M, AGN191183 did not inhibit the TPA-induced Str-AP1-CAT expression. Treatmentwith AGN 193109 in the concentration range of from 10⁻¹⁰ to 10⁻⁸ M didnot substantially inhibit AP-1 mediated reporter activity. However, theresults presented in FIG. 10 indicated that stimulation of thetransfectants with the combination of AGN 193109 (10⁻⁸ M) and AGN 191183in the concentration range of from 10⁻¹² to 10⁻¹⁰ M substantiallyinhibited TPA-induced Str-AP1-CAT expression by an amount of from 12% to21%. Therefore, AGN 193109 potentiated the anti-AP-1 activity of AGN191183 under conditions where this retinoid agonist ordinarily did notinhibit AP-1 activity.

We reasoned that AGN 193109 potentiated the agonist-mediated repressionof AP-1 activity by a mechanism that likely involved AGN193109-dependent sequestration of NCPs onto RARs. RARs belong to asuperfamily of nuclear receptors that also includes receptors for1,25-dihydroxyvitamin D₃, glucocorticoid, thyroid hormone, estrogen andprogesterone. It was a reasonable assumption that the ability to bindNCPs may be shared among different members of the nuclear receptorsuperfamily. This led us to speculate that AGN 193109 could potentiatethe anti-AP-1 activity of one or more of the ligands that interact withthis superfamily of nuclear receptors.

The results presented in the preceding Example clearly indicated thatAGN 193109 potentiated the anti-AP-1 activity of a retinoid agonist.More specifically, AGN 193109 lowered the threshold dose at which theanti-AP-1 activity of AGN 191183 could be detected. Since AGN 193109 hassubstantially no anti-AP-1 activity by itself, its effect on nuclearreceptor agonists was synergistic. We also found that the AGN 193109negative hormone potentiated the anti-AP-1 activity of1,25-dihydroxyvitamin D₃, the natural ligand for the vitamin D₃receptor.

The observed synergy between AGN 193109 and AGN 191183 in the precedingExample necessarily implied that the anti-AP-1 activity of the retinoidagonist and the AGN 193109-mediated potentiation of that activity mustresult from different mechanisms. If the mechanisms of action of the twoagents were identical, then it follows that the effectiveness of thecombination of AGN 193109 and the agonist would have been additive.Instead, the combination was shown to be more effective than eitheragent alone, an effect that could not have been predicted in advance ofthis finding.

Significantly, the AGN 193109-mediated potentiation of the RAR agonistwas performed using an approximately 100-fold molar excess of AGN 193109over that of the retinoid agonist. Accordingly, the majority of RARsshould have been bound by AGN 193109 leaving very few RARs available foragonist binding. In spite of this fact, the population of RARs that werenot bound by AGN 193109 were able to bind retinoid agonist andvigorously stimulate an agonist-dependent response measurable as aninhibition of reporter gene expression. Thus, our data suggestedpossible heterogeneity of RARs that are induced by AGN 193109.

The negative hormone activity of AGN 193109, attributed to its abilityto promote the interaction of RARs and NCPs, provided a basis forunderstanding the synergy between AGN 193109 and retinoid agonists. Ourresults were fully consistent with a model in which AGN 193109 treatmentof cells promoted binding of RARs and NCPs, thereby reducing the numberof free NCP and free RAR within the cell. This results in the generationof two populations of RARs that are functionally distinct. The firstpopulation is represented by RARs associated with NCPs. Such AGN193109/RAR/NCP complexes cannot be activated by retinoid agonists. Thesecond population consists of RARs that are not bound by NCP, and thatremain available for interaction with agonists. This latter populationis designated "RAR*" to indicate free RARs in an environmentsubstantially depleted of NCP.

The RAR*s have decreased probabilities of association with NCP throughequilibrium binding and have an increased sensitivity to retinoidagonists measurable, for example, as anti-AP-1 activity. This is sobecause, while the intracellular reservoir of NCP is depleted by virtueof AGN 193109 administration, the reservoir of PCP has not beendepleted. Accordingly, free RAR*s can bind a retinoid agonist andinteract with PCP factors in an environment substantially depleted ofNCP. The ability of AGN 193109 to increase the sensitivity of othernuclear receptors to their respective agonists can be attributed to theability of these different nuclear receptors to interact with the sameNCPs that interact with AGN 193109/RAR complexes. This model of AGN193109-mediated modulation of NCP availability for nuclear receptorfamily members is schematically represented in FIG. 11.

This mechanistic model led us to predict that AGN 193109 could modulatethe activities of nuclear receptor ligands other than retinoid agonists.As illustrated in the following Example, we confirmed that AGN 193109potentiated the activity of 1,25-dihydroxyvitamin D₃ in an in vitrotransactivation assay.

Example 14 describes the methods used to demonstrate that AGN 193109enhanced the activity of 1,25-dihydroxyvitamin D₃ in a transactivationassay.

Example 14 AGN 193109 Potentiates 1,25-Dihydroxyvitamin D₃ Activity

Hela cells were transfected using the cationic liposome-mediatedtransfection procedure described by Felgner et al. in Proc. Natl. Acad.Sci. USA 84:7413 (1987). 5×10⁴ cells were plated in 12-well multiwellplates and grown in DMEM supplemented with 10% FBS. Cells werecotransfected in serum-free medium using 2 μg/well of LIPOFECTAMINEreagent (Life Technologies, Inc.) with 0.7 μg of the reporter plasmidMTV-VDRE-Luc, containing two copies of the 1,25-dihydroxyvitamin D₃response element 5'-GTACAAGGTTCACGAGGTTCACGTCTTA-3' (SEQ ID NO:4) fromthe mouse osteopontin gene (Ferrara et al. J. Biol Chem. 269:2971(1994)) ligated into the reporter plasmid ΔMTV-Luc (Heyman et al. inCell 68:397 (1992)), and 0.3 μg of the plasmid pGEM3Z (Pharmacia, Inc.)as carrier DNA to bring the final concentration of DNA to 1.0 μg perwell. After six hours of transfection, cells were fed with growth mediumcontaining charcoal extracted FBS at a final concentration of 10%.Eighteen hours after transfection cells were treated with vehicle alone(ethanol) or AGN 193109 in ethanol at a final concentration of either10⁻⁸ or 10⁻⁷ M. Six hours later 1,25-dihydroxyvitamin D₃ was added inethanol to a final concentration of from 10⁻¹⁰ to 10⁻⁷ M. Cells werelysed and harvested eighteen hours following 1,25-dihydroxyvitamin D₃treatment. Luciferase activity was measured as described above. Thisexperimental system allowed a convenient method of monitoring andquantitating 1,25-dihydroxyvitamin D₃ -dependent gene expression.

The results presented in FIG. 12 indicated that, when compared with theresult obtained using 1,25-dihydroxyvitamin D₃ alone, AGN 193109coadministered with 1,25-dihydroxyvitamin D₃ shifted the dose responsecurve to the left. This confirmed that AGN 193109 potentiated theeffectiveness of 1,25-dihydroxyvitamin D₃ in the in vitrotransactivation assay. More specifically, FIG. 12 graphicallyillustrates that an AGN 193109 concentration as low as 10-100 nMrendered the 1,25-dihydroxyvitamin D₃ approximately 10 fold more active.While a 1,25-dihydroxyvitamin D₃ concentration of 10⁻⁸ M was required toproduce a luciferase expression of approximately 2,000 rlu, onlyone-tenth as much 1,25-dihydroxyvitamin D₃ was required to produce thesame luciferase output when the vitamin was coadministered with AGN193109 at a concentration of 10⁻⁸ -10⁻⁷ M. Although not shown on thegraph in FIG. 12, substantially identical results were obtained usingAGN 193109 concentrations of 10⁻⁹ M and 10⁻⁸ M. Thus, coadministrationwith AGN 193109 substantially reduced the amount of1,25-dihydroxyvitamin D₃ that was required to produce a similar effectin the absence of the negative hormone.

Interestingly, when the above procedure was repeated with cotransfectionof a vitamin D receptor (VDR) expression plasmid, there was a coincidentdecrease in the ability of AGN 193109 to potentiate the activity of1,25-dihydroxyvitamin D₃. We interpreted this result as indicating thatover-expression of VDRs could affect the ability of AGN 193109 topotentiate the activity of 1,25-dihydroxyvitamin D₃. Thus, theintracellular concentration of a ligand receptor, which may differ in atissue-specific fashion, can influence the ability of AGN 193109 topotentiate the activity of a ligand that binds the receptor. This wasagain consistent with a model in which titratable NCPs contributed tothe regulation of the Vitamin D₃ response, and supported the model setforth above.

As illustrated in the following Example, we also confirmed that AGN193109 potentiated the anti-AP-1 activity of 1,25-dihydroxyvitamin D₃.Our model for the activity of AGN 193109 action explains thisobservation by invoking that NCPs avidly associate with RARs in thepresence of this drug. Endogenous vitamin D receptors present in HeLacells likely were rendered more sensitive to the 1,25-dihydroxyvitaminD₃ ligand, with the consequence of exaggerating the ability of thisligand to inhibit expression from the Str-AP1-CAT reporter.

Example 15 describes the methods used to demonstrate that AGN 193109potentiated the anti-AP-1 activity of 1,25-dihydroxyvitamin D₃.

Example 15 AGN 193109 Potentiates the Anti-AP-1 Activity of1,25-Dihydroxyvitamin D₃

HeLa cells were transfected with 1 μg of Str-AP1-CAT using LIPOFECTAMINEaccording to the method described by Nagpal et al. in J. Biol. Chem.270:923 (1995). Transfected cells were treated with AGN 193109 alone(10⁻⁹ to 10⁻⁷ M), 1,25-dihydroxyvitamin D₃ alone (10⁻¹² to 10⁻⁷ M) or1,25-dihydroxyvitamin D₃ (10⁻¹² to 10⁻⁷ M) in the presence of 10⁻⁸ M AGN193109.

The results of these procedures indicated that AGN 193109 potentiatedthe ability of 1,25-dihydroxyvitamin D₃ to inhibit TPA-induced AP-1activity. When used alone in the concentration range of from 10⁻⁹ to10⁻⁷ M, AGN 193109 had no detectable anti-AP-1 activity. The resultspresented in FIG. 13 indicated that 1,25-dihydroxyvitamin D₃ repressedTPA-stimulated activity only in the 10⁻⁸ and 10⁻⁷ M concentration range.Analysis of 1,25-dihydroxyvitamin D₃ mediated repression of TPAstimulated CAT activity in the presence of 10⁻⁸ M AGN 193109 indicatedthat anti-AP-1 activity was detectable at 10⁻¹⁰ and 10⁻⁹ M1,25-dihydroxyvitamin D₃ and an increase in activity at 10⁻⁸ and 10⁻⁷ Mdoses compared to 1,25-dihydroxyvitamin D₃ treatment alone. This AGN193109 dependent modulation of 1,25-dihydroxyvitamin D₃ mediatedanti-AP-1 activity was consistent with our model in which NCPsequestration to RARs made the NCP unavailable for interaction withother nuclear receptor family members. Accordingly, the receptors wererendered more sensitive to the 1,25-dihydroxyvitamin D₃ treatment.

The mechanisms underlying RAR mediated transactivation and anti-AP-1activity are likely different. This conclusion was based on ourobservation that high doses of AGN 193109 completely inhibitedtransactivation without substantially inhibiting anti-AP1 activity. Wetherefore wished to gain additional evidence to support our model forRAR* formation mediated by AGN 193109 treatment. To accomplish this, weinvestigated whether AGN 193109 could potentiate the activity of the RARspecific agonist AGN 191183 in an in vitro transactivation assay.

Example 16 describes the methods used to demonstrate that AGN 193109potentiated the activity of the RAR specific agonist, AGN 191183. Theresults of this procedure indicated that, under particularcircumstances, AGN 193109 enhanced the potency of the RAR specificretinoid, and provided strong evidence that AGN 193109 promoted RAR*formation.

Example 16 Potentiation of Retinoid Effectiveness by AGN 193109Coadministration

Hela cells were transfected using the cationic liposome-mediatedtransfection procedure described by Felgner et al. in Proc. Natl. Acad.Sci. USA 84:7413 (1987). 5×10⁴ cells were plated in 12 well multiwellplates and grown in DMEM supplemented with 10% FBS. Cells werecotransfected in serum free medium using LIPOFECTAMINE reagent (2ug/well, Life Technologies, Inc.) with 0.7 μg of the reporter plasmidMTV-TREp-Luc, containing two copies of the TREpal response element5'-TCAGGTCATGACCTGA-3' (SEQ ID NO:5) inserted into the reporter plasmidΔMTV-Luc (Heyman et al. in Cell 68:397 (1992)), and 0.1 μg of the RAR-γexpression plasmid pRShRAR-γ (Ishikawa et al. Mol. Endocrinol. 4:837(1990)). After six hours of transfection, cells were fed with growthmedium containing charcoal extracted FBS at a final concentration of10%. Eighteen hours after transfection, cells were treated with vehiclealone (ethanol) or AGN 193109 in ethanol at a final concentration offrom 10⁻¹¹ to 10⁻⁸ M. Six hours later, AGN 191183 was added in ethanolto a final concentration of either 0, 10⁻¹⁰ or 10⁻⁹ M. Cells wereharvested after eighteen hours of AGN 191183 treatment and luciferaseactivity was measured as described above.

Preliminary experiments indicated that 10⁻⁹ M AGN 193109 was relativelyineffective at inhibiting the response to of 10⁻⁹ M AGN 191183 in HeLacells. This contrasted with the ability of 10⁻⁹ M AGN 193109 to inhibit10⁻⁸ M ATRA in CV-1 cells (FIG. 2).

The results presented in FIG. 14 supported the prediction that AGN193109 stimulated the formation of RAR*. Consistent with ourcharacterization of the antagonist and negative hormone activities ofAGN 193109, treatment with AGN 193109 resulted in a biphasic doseresponse curve. The lowest doses of AGN 193109 (10⁻¹¹ and 10⁻¹⁰ M)resulted in a stimulation of luciferase activity over that of AGN 191183alone. This effect suggests that RAR*s are generated by AGN 193109.Curiously, this was also seen for AGN 193109 treatment alone, suggestingthat RAR*'s can respond to an endogenous ligand. AGN 191183 is asynthetic retinoid agonist and, like ATRA, activates transcriptionthrough the RARs. Substitution of AGN 191183 for ATRA in Example 7 wouldgive qualitatively similar results (i.e., AGN 193109 would antagonizethe effect of 10 nM AGN 191183). Example 16 illustrates that, while AGN193109 can function as an antagonist of RAR agonists, dosing conditionscould easily be identified wherein AGN 193109 coadministrationpotentiated activation mediated by an RAR agonist. It is important tonote that the doses of the compounds used in Example 16 aresubstantially lower than the doses employed in the procedure describedunder Example 7. We proposed that AGN 193109 treatment could lead to RARheterogeneity RARs versus RAR*S. The apparent heterogeneity (i.e.,ability to potentiate) appears to have different windows intransactivation versus AP-1 repression. The reason that the curves arebiphasic is because, with increasing amounts of AGN 193109, there isproportionately less RAR available to bind the agonist. This doesn'tappear to be the case for AP-1 repression and we are left to speculatethat this difference must reflect two distinct mechanisms fortransactivation and AP-1 repression by the same receptor species.

Clinical results have confirmed that some retinoids are useful forinhibiting the growth of premalignant and malignant cervical lesions.Exemplary studies supporting this conclusion have been published byGraham et al. in West. J. Med. 145: 192 (1986), by Lippman et al. in J.Natl. Cancer Inst. 84:241 (1992), and by Weiner et al. in Invest. NewDrugs 4:241 (1986)).

Similar conclusions are supported by the results of in vitro studiesthat used cultured cells to quantitate the antiproliferative effects ofvarious retinoids. More specifically, Agarwal et al. in Cancer Res.51:3982 (1991) employed the ECE16-1 cell line to model the early stagesof cervical dysplasia and demonstrated that retinoic acid could inhibitepidermal growth factor (EGF) dependent cellular proliferation.

Example 17 describes the methods used to demonstrate that AGN 193109 canantagonize the activity of the AGN 191183 retinoid agonist whichinhibited proliferation of the ECE 16-1 cell line.

Example 17 AGN 193109 Antagonizes the Antiproliferative Effect ofRetinoids in ECE16-1 Cells

ECE16-1 cells were seeded at a density of 1×10⁴ cells per cm² incomplete medium containing DMEM:F12 (3:1), nonessential amino acids, 5%FBS, 5 μg/ml transferrin, 2 nM of 3,3',5 triiodothyronine (thyroidhormone or "T₃ "), 0.1 nM cholera toxin, 2 mM L-glutamine, 1.8×10⁻⁴ Madenine and 10 ng/ml EGF. Cells were allowed to attach to platesovernight and then shifted to defined medium containing DMEM:F12 (3:1),2 mM L-glutamine, nonessential amino acids, 0.1% bovine serum albumin,1.8×10⁻⁴ M adenine, 5 μg/ml transferrin, 2 nM T₃, 50 μg/ml ascorbicacid, 100 ug/ml streptomycin, 100 units/ml penicillin and 50 μg/mlgentamicin. Defined medium (DM) was supplemented with 10 ng/ml EGF. EGFtreated cells received 10 nM of the AGN 191183 retinoid agonist incombination with either 0, 0.1, 1.0, 10, 100 or 1000 nM AGN 193109 or1000 nM AGN 193109 alone. After three days of treatment, cells wereharvested as described by Hembree et al. in Cancer Res. 54:3160 (1994)and cell numbers determined using a COULTER counter.

The results presented in FIG. 15 demonstrated that ECE 16-1 cellsproliferated in response to EGF but not in defined medium alone. Thisconfirmed the findings published by Andreatta-van Leyen et al. in J.Cell. Physio. 160:265 (1994), and by Hembree et al. in Cancer Res.54:3160 (1994). Addition of 10 nM AGN 191183 and 0 nM AGN 193109completely inhibited EGF mediated proliferation. Thus, AGN 191183 was apotent antiproliferative retinoid. Increasing the AGN 193109concentration from 0 nM to 10 nM antagonized the AGN 191183 mediatedgrowth inhibition by approximately 50%. A ten-fold molar excess of AGN193109 completely reversed the antiproliferative effect of AGN 191183.Treatment of cells with 1000 nM AGN 193109 alone had no effect on theEGF mediated proliferation increase. These results proved that AGN193109 antagonized the antiproliferative effect of a retinoid but hadsubstantially no antiproliferative activity of its own when used totreat cells representing cervical epithelium that is sensitive to growthinhibition by retinoids such as AGN 191183. Notably, there was noevidence that AGN 193109 potentiated the antiproliferative activity ofthe AGN 191183 agonist using the ECE16-1 model system.

In contrast to the model system represented by the ECE16-1 cell line,there are other examples where cellular proliferation associated withcervical dysplasia cannot be inhibited by retinoid agonists. Forexample, Agarwal et al. in Cancer Res. 54:2108 (1994) described the useof CaSki cells as a model for cervical tumors that are unresponsive toretinoid therapy. As disclosed below, rather than inhibiting cellproliferation, retinoid treatment had substantially no effect on thegrowth rate of CaSki cells. The following Example addressed the effectof the AGN 193109 negative hormone on the proliferation rates of thiscell line. The results unexpectedly proved that AGN 193109 can inhibitthe proliferation of cervical tumor cells that are unresponsive to theantiproliferative effects of retinoid agonists.

Example 18 describes the methods used to demonstrate that AGN 193109inhibited the growth of a cervical tumor cell line that did not respondto the antiproliferative effects of other retinoids such as AGN 191183.Significantly, AGN 193109 displayed antiproliferative activity in theabsence of added retinoid

Example 18 AGN 193109 Inhibits the Proliferation Rate of CaSki CervicalCarcinoma-Derived Cell Line

We tested the effect of EGF on CaSki cell proliferation, either alone orin combination with the AGN 191183 retinoid agonist and/or the AGN193109 negative hormone at a concentration of 10⁻⁶ M. Cell proliferationassays were performed as described above for studies involving ECE16-1cells. EGF was added to the retinoid treated cultures to give a finalconcentration of 20 ng/ml. Cells were treated with AGN 191183 (10⁻¹⁰ to10⁻⁶ M) in the presence or absence of 10⁻⁶ M AGN 193109 for a total ofthree days. The media was replaced with fresh media and each of the tworetinoid compounds, as appropriate, every day. Cell numbers weredetermined using a COULTER counter as described above.

The results presented in FIG. 16 indicated that CaSki cells weresubstantially refractory to the effects of a retinoid agonist and thatAGN 193109 exhibited antiproliferative activity in the absence of addedretinoid. The presence of EGF in the culture media stimulated CaSki cellgrowth. This conclusion was based on comparison of the stripped barrepresenting no AGN 191183 and the open bar representing defined growthmedia ("DM") alone. AGN 191183 treatment had no antiproliferativeactivity on the CaSki tumor cell line. We discounted any slight increasein the cellular proliferation rate associated with the retinoid agonist,because a ten thousand fold increase in the retinoid agonistconcentration was associated with only roughly a 20% increase in theproliferation rate. Thus, the AGN 191183 agonist had substantially noeffect on the proliferation rate of CaSki cells.

The results presented in FIG. 16 also indicated that AGN 193109inhibited proliferation of the CaSki cervical epithelial cell line. Thisconclusion was based on comparison of the measurements appearing as the"0" AGN 191183 black bar and the "0" AGN 191183 stripped bar. Thus, AGN193109 was capable of stimulating a biological response in the absenceof added retinoid agonist when used to treat cervical tumor cells thatwere not growth inhibited by retinoid agonists such as AGN 191183.

Our discovery that the AGN 193109 negative hormone could inhibitcellular proliferation was consistent with a model in which unligandedRAR mediated the expression of genes that were required forproliferation. While an RAR agonist such as AGN 191183 had substantiallyno effect, or perhaps promoted cellular proliferation slightly, AGN193109 had an antiproliferative effect. The AGN 193109 negative hormonelikely bound RARs thereby promoting NCP association and causing the RARsto adopt an inactive conformation. According to our model, thisrepressed gene activity that was positively regulated by unligandedRARs. This ability of AGN 193109 to down-regulate the activity ofunliganded RARs likely resulted from its ability to promote theassociation of RARs and NCPs.

Those having ordinary skill in the art will appreciate that someretinoid agonists are useful for controlling the undesirableconsequences of cell growth that follows retinal detachment. Afterretinal detachment the retinal pigment epithelium (RPE)dedifferentiates, proliferates and migrates into the subretinal space.This process can negatively impact the success of surgical proceduresaimed at retinal reattachment. Campochiaro et al. in Invest. Opthal &Vis. Sci. 32:65 (1991) have demonstrated that RAR agonists such as ATRAexhibited an antiproliferative effect on the growth of primary human RPEcultures. Retinoid agonists have also been shown to decrease theincidence of retinal detachment following retinal reattachment surgery(Fekrat et al. Opthamology 102:412 (1994)). As disclosed in thefollowing Example, we analyzed the ability of the AGN 193109 negativehormone to suppress growth in primary human RPE cultures.

Example 19 describes the methods used to demonstrate that AGN 193109potentiated the antiproliferative effect of a retinoid antagonist in aprimary culture of human retinal pigment epithelium.

Example 19 AGN 193109 Potentiates the Antiproliferative Activity of ATRA

Primary cultures of human retinal pigment epithelium (RPE) wereestablished according to the method described by Campochiaro et al. inInvest. Opthal & Vis. Sci. 32:65 (1991). 5×10⁴ cells were plated in16-mm wells of 24-well multiwell plates in DMEM (Gibco) containing 5%FBS. Cells were mock treated with ethanol vehicle alone, ATRA (10⁻¹⁰ to10⁻⁶ M) in ethanol, AGN 193109 (10⁻¹⁰ to 10⁻⁶ M) in ethanol, or ATRA(10⁻¹⁰ to 10⁻⁶ M) and 10⁻⁶ M AGN 193109. Cells were fed with fresh mediacontaining the appropriate concentrations of these compounds every twodays for a total of five days of treatment. Cells were removed from theplates by gentle digestion with trypsin and the number of cells wascounted with an electronic cell counter.

The results presented in FIG. 17 indicated that AGN 193109 dramaticallypotentiated the antiproliferative activity of ATRA on RPE cells.Treatment of primary RPE cells with ATRA led to a dose dependentdecrease in RPE cell proliferation with an approximately 40% decrease at10⁻⁶ M ATRA relative to control cultures. AGN 193109 treatment did notsubstantially alter the growth rate of the RPE cells at anyconcentration tested in the procedure. Unexpectedly, the combination ofATRA (10⁻¹¹ to 10⁻⁶ M) and 10⁻⁶ M AGN 193109 had a strongerantiproliferative activity than ATRA alone. Thus, AGN 193109 cotreatmentpotentiated the antiproliferative effect of ATRA. More specifically, theresults shown in the Figure indicated that the antiproliferative effectof 10⁻⁸ M ATRA was obtainable using only 10⁻¹⁰ M ATRA in combinationwith 10⁻⁷ M AGN 193109. Thus, the AGN 193109 negative hormoneadvantageously enhanced the antiproliferative activity of ATRA byapproximately 100 fold.

In an independent experiment, comparison of the antiproliferative effectof ATRA (10⁻¹¹ to 10⁻⁶ M) with that of ATRA and 10⁻⁶ M AGN 193109 againdemonstrated the apparent increase in sensitivity of primary RPE cellsto ATRA in the presence of AGN 193109. In this system, AGN 193109neither functioned as a retinoid antagonist nor exhibited anantiproliferative effect when used alone. However, AGN 193109coadministration potentiated the antiproliferative activity of theretinoid agonist.

AGN 193109 was tested for its ability to potentiate theanti-proliferative effect of 13-cis retinoic acid (13-cis RA) in primaryRPE cultures using conditions and techniques to measure RPE cellproliferation described above. Notably, 13-cis RA is clinicallysignificant. More particularly, 13-cis RA is useful in the treatment ofseveral disease states, including acne (Peck et al. N. Engl. J. Med.300:329 (1977); Jones et al. Br. J. Dermatol. 108:333 (1980)), andsquamous cell carcinoma of the skin and cervix in combination treatmentwith interferon 2α (Lippman et al. J. Natl. Cancer Inst. 84:241 (1992);Moore et al. Seminars in Hematology 31:31 (1994)).

The results presented in FIG. 18 indicated that both 13-cis RA (10⁻¹² to10⁻⁶ M) and ATRA (10⁻¹² to 10⁻⁶ M) effectively inhibited RPE cellgrowth. Notably, the 13-cis isomer was approximately two orders ofmagnitude less effective in this assay when compared with ATRA. Similarto the results obtained using coadministration of AGN 193109 and ATRA(above), coadministration of AGN 193109 (either 10⁻⁸ or 10⁻⁶ M) with13-cis RA (10⁻¹² to 10⁻⁶ M) dramatically increased the potency of 13-cisRA in mediating repression of RPE cell proliferation. In contrast totreatment with 13-cis RA alone, coadministration of AGN 193109 enhancedthe potency of 13-cis RA. Thus, AGN 193109 potentiated theantiproliferative activity of 13-cis RA.

We next tested the ability of AGN 193109 to potentiate the activities ofother nuclear receptor hormones in primary RPE cell cultures.Dexamethasone, a synthetic glucocorticoid receptor agonist, is onemember of a class of compounds that have been used clinically for theirpotent anti-inflammatory and immunosuppressive properties. Thyroidhormone (T3; 3,3',5'-Triiodothyronine) is a natural thyroid hormonereceptor agonist used primarily for hormone replacement therapy in thetreatment of hypothyroidism. Methods used in these experiments wereidentical to those described above for procedures employing ATRA and13-cis RA.

The results of these procedures indicated that coadministration of AGN193109 and the nuclear receptor agonists potentiated theantiproliferative activities of the nuclear receptor agonists. Morespecifically, the results presented in FIG. 19 showed that single-agenttreatment of RPE cells with either dexamethasone (10⁻¹¹ to 10⁻⁶ M) orATRA (10⁻¹² to 10⁻⁶ M) was substantially unable to inhibit RPE cellproliferation. However, treatment of RPE cells with dexamethasone (10⁻¹¹to 10⁻⁶ M) and either 10⁻⁸ or 10⁻⁶ M AGN 193109 repressed RPE cellproliferation to an extent that approximated the inhibition caused bytreatment with ATRA. Similarly, the results presented in FIG. 20indicated that AGN 193109 potentiated the antiproliferative activity ofthyroid hormone. Similar to the results obtained using dexamethasone,the proliferation of RPE cells was refractory to single-agent treatmentwith thyroid hormone (10⁻¹¹ to 10⁻⁶ M). However, co-treatment of RPEcells with thyroid hormone (10⁻¹¹ to 10⁻⁶ M) and AGN 193109 (either 10⁻⁸or 10⁻⁶ M) inhibited RPE cell proliferation in a thyroid hormonedependent manner. We concluded that AGN 193109 rendered primary RPEcultures sensitive to the anti-proliferative effects of these nuclearreceptor agonists. The mechanism by which AGN 193109 mediated theseeffects likely involved modulation of NCP/RAR interactions.

We additionally examined the effect of AGN 193109 on the expression ofmarker genes in other experimental systems that were sensitive toretinoid agonists. Both the MRP8 and stromelysin genes are known to beinhibited by retinoid agonists in a variety of biological systems. Forexample, Wilkinson et al. in J. Cell Sci. 91:221 (1988) and Madsen etal. in J. Invest. Dermatol. 99:299 (1992) have disclosed that MRP8 geneexpression was elevated in psoriasis. Conversely, MRP8 gene expressionwas repressed by the retinoid agonist AGN 190168 in human psoriatic skin(Nagpal et al., submitted 1995), in human keratinocyte raft cultures(Chandraratna et al. J. Invest. Dermatol. 102:625 (1994)) and incultured human newborn foreskin keratinocytes (Thacher et al. J. Invest.Dermatol. 104:594 (1995)). Nagpal et al. in J. Biol. Chem. 270:923(1995) have disclosed that stromelysin mRNA levels were repressed byretinoid agonists such as AGN 190168 in cultured human newborn foreskinkeratinocytes. We analyzed the regulated expression of these genesfollowing treatment of cultured human newborn foreskin keratinocyteswith either the AGN 191183 retinoid agonist or AGN 193109.

Example 20 describes the methods used to demonstrate that AGN 193109inhibited MRP-8 expression in cultured keratinocytes.

Example 20 AGN 193109 Inhibits MRP-8 Expression in Keratinocytes

Primary foreskin keratinocytes were isolated according to the proceduredescribed by Nagpal et al. in J. Biol. Chem. 270:923 (1995) and culturedin keratinocyte growth medium (KGM) that was purchased from Clonetics.After 3 days of treatment with AGN 191183 (10⁻⁷ M) or AGN 193109 (10⁻⁶M), total cellular RNA was isolated from treated and controlkeratinocytes according to standard methods. The mRNA was reversetranscribed into cDNA which then served as the template in a PCRamplification protocol using primers specific for either theglyceraldehyde phosphate dehydrogenase (GAPDH) housekeeping gene orMRP-8. The GAPDH primers had the sequences5'-CCACCCATGGCAAATTCCATGGCA-3' (SEQ ID NO:6) and5'-TCTAGACGGCAGGTCAGGTCCACC-3' (SEQ ID NO:7). The MRP-8 primers had thesequences 5'-ACGCGTCCGGAAGACCTGGT-3' (SEQ ID NO:8) and5'-ATTCTGCAGGTACATGTCCA-3' (SEQ ID NO:9). An aliquot from the MRP-8amplification reaction (10 μl) was removed after every cycle of PCRamplification starting from 12 cycles and ending at 21 cycles.Similarly, an aliquot of the GAPDH amplification reaction was removedafter every PCR cycle starting at 15 cycles and ending at 24 cycles. Thesamples were electrophoresed on 2% agarose gels and the separatedamplification products detected by ethidium bromide staining. Thestaining intensity of the amplification products served as aquantitative measure of the amount of starting mRNA specific for thegiven primer set.

The results of this procedure indicated that both AGN 191183 and AGN193109 independently inhibited MRP-8 expression in keratinocytes. Theintensity of the stained GAPDH amplification product was substantiallyequivalent in the lanes of the gel representing starting materialisolated from control, AGN 191183, and AGN 193109 treated keratinocytes.Weak bands representing the GAPDH amplification product were firstdetectable in lanes corresponding to samples removed after 18 cycles ofPCR amplification. The equivalent staining intensities among the variouslanes of the gel indicated that equivalent masses of starting materialwere used for all samples. Accordingly, differences in the intensitiesof stained bands representing MRP-8 amplification products wereindicative of differences in MRP-8 mRNA expression among the variousstarting samples. As expected, the MRP-8 amplified signal was inhibitedin AGN 191183 (10⁻⁷ M) treated cultures relative to an untreatedcontrol. AGN 193109 (10⁻⁶ M) treatment of cultured keratinocytes alsorepressed MRP8 expression as judged by lower intensity of stainedamplification product.

As illustrated in the following Example, AGN 193109 also inhibitedexpression of a second marker gene in keratinocytes. Nagpal et al. in J.Biol. Chem. 270:923 (1995) disclosed that stromelysin mRNA expressionwas down-regulated by RAR specific agonists in cultured newborn humanforeskin keratinocytes. Nicholson et al. (EMBO J. 9:4443 (1990))disclosed that an AP-1 promoter element played a role in theretinoid-dependent negative regulation of the stromelysin-1 gene. Thus,it was of interest to determine whether AGN 193109 could alter theexpression of this gene.

Example 21 describes the methods used to demonstrate that AGN 193109inhibited stromelysin-1 gene expression in the absence of an exogenouslyadded retinoid agonist.

Example 21 AGN 193109 Inhibits Stromelysin-1 Expression in CulturedKeratinocytes

Primary foreskin keratinocytes were either mock treated or treated for24 hours with the RAR agonist AGN 191183 (10⁻⁷ M), or AGN 193109 (10⁻⁶M). Total RNA prepared from mock-treated and retinoid-treatedkeratinocytes was reverse transcribed and the resulting cDNA was PCRamplified using β-actin or stromelysin-1 oligo primers exactly asdescribed by Nagpal et al. in J. Biol. Chem. 270:923 (1995)), thedisclosure of which has been incorporated by reference. A sample (10 μl)from the PCR amplification reaction was removed after every three cyclesstarting from 18 cycles of PCR amplification. The sample waselectrophoresed on a 2% agarose gel and detected after ethidium bromidestaining.

Results of these procedures indicated that AGN 193109 inhibitedstromelysin-1 gene expression in the absence of an exogenously addedretinoid agonist. More specifically, ethidium-stained bands representingβ-actin amplification products were easily detectable the agarose gelsafter 18 cycles of PCR. While all band intensities increased withadditional cycles of the amplification reaction, stained bands weresomewhat less intense in samples representing AGN 191183 treated cells.This indicated that a slightly lesser amount of RNA must have beenpresent in the starting samples corresponding to cells treated with AGN191183. The results also indicated that stromelysin-1 mRNA wasdetectable in mock-treated keratinocytes starting at 33 cycles of PCRamplification. As expected, stromelysin-1 mRNA expression was inhibitedafter AGN 191183 (10⁻⁷ M) treatment as judged by the weaker bandintensity on when compared with samples derived from mock-treatedsamples. When normalized to the intensities of the β-actin amplificationproducts, and consistent with the results obtained in measurements ofMRP-8 expression, AGN 193109 (10⁻⁶ M) treatment of keratinocytesresulted in down-regulation of stromelysin-1 mRNA levels. Indeed, thedown-regulation stimulated by AGN 193109 treatment was indistinguishablefrom the down-regulation caused by treatment of keratinocytes with theRAR agonist AGN 191183.

As disclosed herein, AGN 193109 can have any of three possible effectswith respect to modulating the activity of a coadministered steroidsuperfamily agonist. First, AGN 193109 may have no effect. Second, AGN193109 may antagonize the effect of the agonist, thereby leading to adecrease in the activity of the agonist. Finally, AGN 193109 maypotentiate the activity of the agonist, thereby leading to a stimulationof the measured effect produced by the agonist.

Compounds having activities that can be modulated by AGN 193109 includeretinoid receptor agonists and agonists which bind to other members ofthe steroid receptor superfamily. This latter category of agonistsincludes vitamin D receptor agonists, glucocorticoid receptor agonistsand thyroid hormone receptor agonists. Peroxisome proliferator-activatedreceptors, estrogen receptor and orphan receptors having presentlyunknown ligands may also be potentiated by AGN 193109. In the case wherethe steroid superfamily agonist is an RAR agonist, AGN 193109 may eitherantagonize or potentiate the activity of that agonist. In the case wherethe agonist used in combination with AGN 193109 is a compound that canbind to a nuclear receptor other than an RAR, coadministration of AGN193109 will either have no effect or will sensitize of the system to theagonist so that the activity of the agonist is potentiated.

A generalized exemplary procedure for determining which of the threepossible activities AGN 193109 will have in a particular system follows.This description illustrates each of the possible outcomes for AGN193109 coadministration with a steroid receptor superfamily agonist.Biological systems useful for assessing the ability of AGN 193109 tomodulate the activity of a nuclear receptor agonist include but are notlimited to: established tissue culture cell lines, virally transformedcell lines, ex-vivo primary culture cells and in vivo studies utilizingliving organisms. Measurement of the biological effect of AGN 193109 insuch systems could include determination of any of a variety ofbiological endpoints. These endpoints include: analysis of cellularproliferation, analysis of programmed cell death (apoptosis), analysisof the differentiation state of cells via gene expression assays,analysis of the ability of cells to form tumors in nude mice andanalysis of gene expression after transient or stable introduction ofreporter gene constructs.

For illustrative purposes, an mRNA species designated as mRNA "X" isexpressed from gene "X" in primary cultured "Y" cells isolated from theorgan "Z." Under standard culture conditions, where several "Y" cellgenetic markers are maintained, including expression of gene "X",addition of a retinoid agonist leads to a decrease in the abundance of"X" mRNA. Analysis of gene X expression can be assessed via isolation ofcellular mRNA and measurement of the abundance of X mRNA levels viapolymerase chain reaction, ribonuclease protection or RNA blottingprocedures such as Northern analyses. After isolation from organ Z,primary Y cells are cultured in an appropriate growth medium. Theprimary cultures are then plated into tissue culture plates forexpansion of the cell population. This step facilitates separation ofthe cells into four sample groups so that various doses of the retinoidagonist and AGN 193109 can be delivered. The first group will be acontrol, receiving vehicle only. The second group will receive the RARagonist, retinoic acid, delivered in ethanol, in amounts sufficient toprovide final concentrations in the range of from 10⁻¹¹ to 10⁻⁶ M. Thelowest dose may need to be empirically determined depending on thesensitivity of the system. Such determinations fall within the scope ofroutine experimentation for one having ordinary skill in the art. Thethird group will receive both the nuclear receptor agonist at the samedoses used for treating the cells of group 2, and a constant dose of AGN193109. The dose of AGN 193109 used for treating the cells of group 3will also need to be determined empirically, but should approximate theaffinity constant (Kd) of AGN 193109 for the RAR subtypes (i.e., atleast 10⁻⁸ M). The fourth group will receive AGN 193109 at dosesminimally including that used for agonist coadministration in group 3.An alternative to this dosing regimen would substitute AGN 193109 forthe retinoid agonist described in the foregoing example, as specified ingroup 2, and a constant dose of retinoid agonist in place of AGN 193109,as specified in groups 3 and 4. After a suitable incubation period,cells should be harvested in a manner suitable for determination of thebiological endpoint being measured as an indicator of agonist activity.

For example, analysis of the effect of AGN 193109 on retinoic aciddependent regulation of gene expression would involve comparison of theabundance of the mRNA species X in the mRNA pool harvested from cellstreated according to each of the four protocols described above. RNAderived from control cells will serve to determine the baselineexpression of X mRNA and will represent a condition corresponding to norepression. Comparison of this level with that measured in the mRNA poolderived from cells treated with retinoic acid will allow fordetermination of the effect of this agonist on gene expression.Quantitated levels of the repression of specific mRNAs resulting fromretinoic acid treatment can then be compared with mRNA abundances fromcells treated in parallel with either AGN 193109 alone or AGN 193109 incombination with retinoic acid. While this generalized exampleillustrates an analysis of the effect of coadministered AGN 193109 onthe expression of a gene repressed by a retinoid agonist, the examplecould alternatively have described analysis of the effect ofcoadministered AGN 193109 on a gene that was induced by a retinoidagonist. The critical feature for determining whether AGN 193109 willbehave as an agonist, as a negative hormone or have no effect in aparticular system will involve quantitative comparison of the magnitudeof the effect in the presence and absence of AGN 193109.

An example in which AGN 193109 potentiated the activity of acoadministered agonist would be a case in which AGN 193109 cotreatmentwith retinoic acid resulted in a level of X mRNA expression that isfurther repressed relative to the level measured in cells treated withretinoic acid alone. More specifically, comparison of the dose responsecurve of the biological effect (i.e., repression of X mRNA abundance)plotted on the Y-axis versus the dose of the agonist (logarithmic scale)on the X-axis would allow comparison of agonist-mediated repression of XmRNA abundance in the presence and absence of AGN 193109 cotreatment.The ability of AGN 193109 to sensitize the biological response to theagonist, thereby potentiating the activity of the agonist, will beindicated by a leftward shift in the dose response curve. Morespecifically, in the presence of AGN 193109 less agonist would berequired to obtain the same biological effect obtainable using theagonist alone.

An example of AGN 193109 mediating antagonism of a coadministeredagonist would be a case in which AGN 193109 cotreatment with retinoicacid resulted in a level of X mRNA expression that is less repressedcompared to that measured in cells treated with retinoic acid alone.Comparison of dose response curves of X mRNA repression versus log doseof agonist in the presence and absence of AGN 193109 will demonstrate ashift to the right in the dose response curve. More specifically, in thepresence of AGN 193109, more agonist will be necessary to obtain thesame biological effect obtainable with single agent treatment with theagonist alone.

The above examples wherein AGN 193109 mediates either antagonism orpotentiation describe experimental outcomes for coadministration of AGN193109 with a retinoid agonist. If, however, the agonist coadministeredwith AGN 193109 is an agonist capable of binding and activating a memberof the steroid receptor superfamily other than an RAR, then instead ofantagonizing the agonist, it becomes possible that AGN 193109 would haveno effect on the activity of the agonist. If AGN 193109 cotreatment withsuch an agonist results in a level of mRNA expression which is equal tothat measured in cells treated with agonist alone, then AGN 193109'sability to affect the availability of NCPs via promotion of RAR:NCPassociations will be silent in this system. This would be an examplewherein AGN 193109 has no effect on a coadministered agonist.

Example of Antagonism

The method disclosed in the above generalized example for determiningthe effect of AGN 193109 coadministered with a retinoid agonist isexemplified by the procedure described under Example 7. CV-1 cellscotransfected with one of the three retinoic acid receptors and theretinoid agonist inducible MTV-TREp-Luc reporter construct were dosedwith either ethanol (control, group 1), AGN 193109 at finalconcentrations of from 10⁻⁹ to 10⁻⁶ M (group 2), AGN 193109 at finalconcentrations of from 10⁻⁹ to 10⁻⁶ M coadministered with retinoic acidat 10⁻⁸ M (group 3), or retinoic acid (10⁻⁸ M, group 4). Comparison ofthe luciferase activity of group 1 with that of group 4 alloweddetermination of the level of retinoid agonist induced expression of theluciferase reporter gene in the absence of added AGN 193109. Comparisonof luciferase reporter gene expression in cells of group 3 with thatmeasured in cells of group 4 indicated that AGN 193109 behaved as anantagonist of the retinoid agonist in this system.

Example of Antagonism

The method disclosed in the generalized example for determining theeffect of AGN 193109 coadministered with a retinoid agonist wassimilarly used to determine in Example 17 that AGN 193109 functioned asan antagonist of a retinoid agonist-mediated repression ofEGF-stimulated cellular proliferation in ECE-16-1 transformed cervicalepithelial cells. In this procedure, treatments of ECE-16-1 cellsincluded a control sample treated with EGF alone (group 1), a sampletreated with the combination of EGF and AGN 193109 at a finalconcentration of 10⁻⁶ M (group 2), a sample treated with the combinationof EGF and AGN 193109 at final concentrations of from 10⁻¹⁰ to 10⁻⁶ Mcoadministered with a single dose of the retinoid agonist AGN 191183 at10⁻⁸ M (group 3), and a sample treated with the combination of EGF andAGN 191183 at 10⁻⁸ M (group 4). After three days of treatment, cellularproliferation rates were determined. Determination that the cells hadbeen stimulated to proliferate by EGF was possible because an additionalcontrol treatment was included wherein cells were exposed to definedmedium that did not contain EGF. Comparison of the number of cells ingroup 1 with the number of cells in group 4 allowed for determinationthat RAR agonist AGN 191183 repressed the EGF-stimulated proliferationof ECE-16-1 cells. Comparison of group 3 with group 4 indicated that AGN193109 antagonized the activity of the RAR agonist in this system.

Example of Potentiation

The method disclosed in the generalized example for determining theeffect of AGN 193109 coadministered with a retinoid agonist was alsoused in Example 14 to determine that AGN 193109 potentiated the activityof a nuclear receptor agonist in HeLa cells transfected with the1,25-dihydroxyvitamin D₃ inducible MTV-VDRE-Luc reporter gene.Treatments of transfected cells included vehicle alone (control, group1), 1,25-dihydroxyvitamin D₃ at final concentrations of from 10⁻¹⁰ to10⁻⁷ M (group 2), 1,25-dihydroxyvitamin D₃ at final concentrations offrom 10⁻¹⁰ to 10⁻⁷ M coadministered with AGN 193109 at a finalconcentration of either 10⁻⁸ or 10⁻⁷ M (group 3), and AGN 193109 as asingle agent treatment at a final concentration of either 10⁻⁸ or 10⁻⁷ M(group 4). Comparison of the luciferase activity measured in group 1(control) cells with that of group 2 cells allowed for determinationthat 1,25-dihydroxyvitamin D₃ stimulated luciferase activity wasdose-dependent. Comparison of luciferase activity measured in cells ofgroup 4 (AGN 193109 single agent treatment) with that measured in cellsof group 3 (AGN 193109 coadministration) similarly allowed fordetermination of dose-dependent 1,25-dihydroxyvitamin D₃ stimulatedluciferase activity in the presence of a given concentration of AGN193109. In this instance, the zero value represented the luciferaseactivity in cells treated with AGN 193109 alone (group 4). Such a dosingregimen allowed for comparison of three 1,25-dihydroxyvitamin D₃ doseresponse curves. Comparison of the dose response curve of1,25-dihydroxyvitamin D₃ in the absence of AGN 193109 with the curverepresenting coadministration of AGN 193109 (either 10⁻⁸ or 10⁻⁷ M)demonstrated potentiation of the agonist activity as evidenced by aleftward shift in the half-maximal response.

Example of Potentiation

The method disclosed in the generalized example for determining theeffect of AGN 193109 coadministered with a retinoid agonist was furtherused to determine in Example 19 that AGN 193109 potentiated theantiproliferative activity of an RAR agonist in primary cultures ofhuman retinal pigment epithelium cells. Treatments of cells included:ethanol vehicle alone (group 1), retinoic acid at final concentrationsof from 10⁻¹⁰ to 10⁻⁶ M (group 2), retinoic acid at final concentrationsof from 10⁻¹⁰ to 10⁻⁶ M coadministered with 10⁻⁶ M AGN 193109 (group 3),and AGN 193109 alone at final concentrations of from 10⁻¹⁰ to 10⁻⁶ M(group 4). Comparison of assay results obtained using cells of groups 1and 2 allowed for determination of the dose dependent inhibition ofproliferation of these cells by retinoic acid. Similarly, comparison ofresults obtained using cells of group 3 with those of group 1 allowedfor determination of the dose dependent inhibition of proliferation ofthese cells by retinoic acid in the presence of coadministered AGN193109. Group 4 demonstrated the inability of AGN 193109 tosubstantially alter the proliferation rate of these cells when used as asingle treatment agent. Comparison of the dose response curves ofretinoic acid mediated repression of cellular proliferation generated ingroups 2 and 3 provided the basis for the conclusion that AGN 193109sensitized primary RPE cells to the antiproliferative effects of the RARagonist, thereby potentiating the activity of the RAR agonist.

As indicated above, Agarwal et al., in Cancer Res. 54:2108 (1994)),showed that CaSki cell growth, unlike the growth of HPV immortalizedECE-16-1 cells, was not inhibited by treatment with retinoid agonists.As disclosed herein, we unexpectedly found that CaSki cell growth wasinhibited by AGN 193109 in the absence of a retinoid agonist. Thefollowing Example illustrates how AGN 193109 can be used to inhibit thegrowth of CaSki cell tumors in vivo.

Example 22 Inhibition of CaSki Cell Tumor Growth in Nude Mice FollowingAdministration of AGN 193109

1×10⁶ CaSki cells are injected into each of a panel of nude mice. Tumorformation is assessed using techniques that will be familiar to onehaving ordinary skill in the art. After injection, mice are randomlydivided into control and test groups. The control group receives aplacebo. The test group is administered with AGN 193109. Animalsadministered with the placebo receive intragastric intubation of cornoil. The test animals receive 20 μMol/kg AGN 193109 in corn oil dailyfor the period of the treatment. Tumor volume is measured in cubicmilliliters using graduated calipers. Tumor volume is plotted asfunction of time. Mice receiving AGN 193109 exhibit tumors which aresignificantly reduced in their growth rate as compared to tumors incontrol mice as judged by tumor size and number over the period of thestudy. This result provides an in vivo demonstration that AGN 193109inhibits the growth of an advanced cervical carcinoma that is resistantto therapy comprising administration of a retinoid agonist.

As indicated above, CaSki cells are a model of cervical tumors that arenot responsive to retinoid agonist therapy. However, herein we havedisclosed that CaSki cell growth was inhibited by AGN 193109 in theabsence of treatment with a retinoid agonist. The ability of AGN 193109to inhibit the proliferation of CaSki cells suggested that AGN 193109could be used to therapeutically treat cervical carcinomas that areinsensitive to retinoid agonist therapy. The following Exampleillustrates one method that can be used to assess the therapeuticpotential of AGN 193109 in the treatment of a cervical carcinoma.

Example 23 Assessing the Therapeutic Potential of AGN 193109 in PatientsHaving Cervical Carcinoma

A patient presenting with an advanced cervical carcinoma is firstidentified. A cervical biopsy is obtained according to methods that willbe familiar to one having ordinary skill in the art. Cells from theexplanted tumor are propagated in tissue culture according to standardtechniques to provide cell numbers sufficient to allow division intothree sample groups. Culture conditions described by Agarwal et al. inCancer Res. 54:2108 (1994) are employed for this purpose. The firstgroup is reserved as a control and receives vehicle alone (ethanol). Thesecond group is treated with the RAR agonist retinoic acid at aconcentration of from 10⁻¹⁰ to 10⁻⁶ M. The third group is treated withAGN 193109 at doses ranging from 10⁻¹⁰ to 10⁻⁶ M. Cells are fed withfresh growth medium daily and are provided with the retinoids describedabove as appropriate for each sample group. Cells are counted afterthree days using an electric cell counter. Comparison of the number ofcells in control cultures with the number of cells in retinoic acidtreated cultures indicates the RAR agonist does not substantiallyinhibit the growth rate of the cultured cervical carcinoma cells. Incontrast, cells treated with AGN 193109 exhibit a dose-dependentdecrease in cell number when compared with cell counts in the controlgroup. This result, wherein AGN 193109 treatment inhibits culturedcervical carcinoma cell proliferation, indicates that AGN 193109 will bea useful therapeutic agent for treating cervical carcinoma patientshaving metastatic disease.

Cervical carcinoma patients having undergone surgery for the removal ofprimary tumors and who present with metastatic disease are enlisted in arandomized clinical study seeking to demonstrate the therapeutic benefitof AGN 193109 in this indication. Patients are divided into two groups.The first group is a control group while members of the second group aretreated with AGN 193109. AGN 193109 is combined with a pharmaceuticallyacceptable excipient to produce a composition suitable for systemicadministration, all according to techniques that will be familiar to onehaving ordinary skill in the art. The control group is administered aplacebo formulation and the experimental group is administered with theformulation containing the AGN 193109 negative hormone. Dosing ofpatients is at the maximum tolerated dose and is performed every otherday for a period of from three months to one year. The outcome of thestudy is quantified via measurement of disease-free survival over time.Individuals receiving AGN 193109 display a significant increase indisease-free survival, including a disproportionate number of patientsdisplaying complete remission of their metastatic disease. This resultindicates that AGN 193109 has therapeutic utility for in vivo treatmentof cervical carcinomas that are unresponsive to the antiproliferativeeffects of retinoid agonists, such as retinoic acid.

As disclosed above, AGN 193109 potentiated the antiproliferativeactivity of RAR agonists in primary cultures of human retinal pigmentepithelium cells. Accordingly, coadministration of AGN 193109 with anRAR agonist in vivo is reasonably expected to increase the therapeuticindex of the agonist because a lesser amount of the RAR agonist will berequired to obtain the same therapeutic endpoint. Additionally, AGN193109 has been demonstrated to sensitize primary cultures of humanretinal pigment epithelium cells to the antiproliferative effects ofglucocorticoid and thyroid hormone receptor agonists. The followingrabbit model of PVR will be utilized in two separate studies todemonstrate the increased therapeutic index obtained viacoadministration of AGN 193109 with an RAR agonist (13-cis retinoicacid) or a thyroid hormone receptor agonist, respectively. Notably, therabbit model of retinal redetachment published by Sen et al. in Arch.Opthalmol. 106:1291 (1988), has been used to demonstrate that retinoidagonists which inhibit proliferation of primary RPE cells in vitro alsoinhibit the frequency of retinal detachment in vivo (Araiz et al.Invest. Opthalmol. 34:522 (1993)). Thus, with respect to their use astherapeutics in the prevention of retinal detachment, a correlationbetween the in vitro and in vivo activities of retinoid agonists hasalready been established. The following Examples illustrate how AGN193109 can be used in therapeutic applications directed at preventingretinal detachment.

Example 24 Use of AGN 193109 to Increase the Therapeutic Potential ofSteroid Superfamily Receptor Agonists in the Treatment of ProliferativeVitreoretinopathy (PVR)

In a first study, human RPE cells are injected into the vitreous cavityof rabbit eyes according to the method described by Sen et al. in Arch.Opthalmol. 106:1291 (1988). After intravitreal injection, the rabbitsare divided into five groups. The first group (control) will receivevehicle alone by intravitreal injection. The second group receivesretinoic acid as single agent treatment (100 μg) by intravitrealinjection. The third group receives AGN 193109 as a single agenttreatment (100 μg) by intravitreal injection. The fourth group receivesby intravitreal injection the RAR agonist (retinoic acid) at a doseone-tenth the amount administered to group 2 (10 μg). The fifth groupreceives the combination of AGN 193109 (100 μg) and retinoic acid (10μg) by intravitreal injection. Animals receive a single intravitrealinjection of the appropriate treatment one day after intravitrealinjection of human RPE cells. Rabbits are examined by indirectophthalmoscopy on days 7, 14 and 28, and are graded for the frequencyand severity of tractional retinal detachment. Rabbits from the groupinjected with 100 μg retinoic acid exhibit a significantly reducedfrequency and severity of retinal detachment compared to control rabbitsor rabbits receiving either AGN 193109 or retinoic acid (10 μg) alone.Rabbits in the group administered with the combination of AGN 193109 andretinoic acid (10 μg) exhibit significantly reduced frequency andseverity of retinal detachment as compared to those in groups eithercontrol, AGN 193109 or retinoic acid (10 μg). This result demonstratesthat AGN 193109 improves the therapeutic index of the RAR agonistretinoic acid in an in vivo model of PVR.

In a second study, rabbits are first provided with an injection of humanRPE cells into the vitreous cavity of the eye, and then divided intofour groups. The first group (control) receives vehicle alone byintravitreal injection. The second group receives thyroid hormone assingle agent treatment (100 μg) by intravitreal injection. The thirdgroup is administered with AGN 193109 as a single agent treatment (100μg) by intravitreal injection. The fourth group is administered with thecombination of AGN 193109 (100 μg) and thyroid hormone (100 μg). Rabbitsare examined by indirect ophthalmoscopy on days 7, 14 and 28, and gradedfor the frequency and severity of tractional retinal detachment.Comparison of the frequency and severity of retinal detachment in thefour groups demonstrates that single agent treatment with either AGN193109 or thyroid hormone does not inhibit retinal detachment whencompared with control rabbits. In contrast, the group of rabbitsadministered with the combination of AGN 193109 and thyroid hormoneexhibit significantly reduced incidence and severity of retinaldetachment. This result demonstrates that AGN 193109 improves thetherapeutic index of thyroid hormone in an in vivo model of PVR.

The following Example illustrates how AGN 193109 can be used to enhancethe therapeutic index of an RAR agonist used to treat human patientsfollowing retinal reattachment surgery.

Example 25 Increasing the Therapeutic Index of RAR Agonist 13-cisRetinoic Acid

A population of adult volunteers having retinal detachment resultingfrom PVR is first identified. Individuals undergo surgical repair of thedetachments using techniques that are standard in the art. The patientsare then divided into five groups. The control group consists ofpatients who undergo surgical repair of the retinal detachment and donot receive any retinoid compound. The second group receives 40 mg oral13-cis retinoic acid twice daily for four weeks postoperatively. Thethird group receives 40 mg oral AGN 103109 twice daily for four weekspostoperatively. The fourth group receives 4 mg oral 13-cis retinoicacid twice daily for four weeks postoperatively. The fifth groupreceives 40 mg oral AGN 193109 in combination with 4 mg oral 13-cisretinoic acid twice daily for four weeks postoperatively. The treatmentprotocol and assessment of drug efficacy is performed essentially asdescribed by Fekrat et al. in Ophthalmology 102:412 (1995).

The frequency and severity of retinal redetachment in postoperativepatients in all five groups is monitored over a period of nine monthsusing ophthalmologic examination techniques that will be familiar tothose of ordinary skill in the art. Patients receiving 40 mg oral 13-cisretinoic acid exhibit significantly reduced incidence of retinalredetachment when compared with control patients, patients receiving 4mg oral 13-cis retinoic acid twice daily or patients receiving 40 mgoral AGN 193109 twice daily. Examination of the patient group receivingthe combination of 40 mg oral AGN 193109 and 4 mg oral 13-cis retinoicacid twice daily for four weeks postoperatively demonstrates thetherapeutic outcome in this patient group is equal to or better thanthose patients receiving 40 mg oral 13-cis retinoic acid twice daily forfour weeks postoperatively. This result demonstrates that the AGN 193109negative hormone improves the therapeutic index of an RAR agonist byvirtue of decreasing the frequency and severity of retinal redetachmentin PVR patients.

Generalized Assay for Identifying Nuclear Receptor Negative Hormones

We have demonstrated above that AGN 193109 can function as a negativehormone capable of repressing the basal transcriptional activity of RARnuclear receptors. Further, we have described an assay using CV-1 cellsco-transfected with the ERE-tk-Luc luciferase reporter plasmid and theER-RXR-α and RAR-γ-VP-16 receptor expression plasmids for distinguishingRAR ligands that are simple antagonists from those having negativehormone activity.

We have concluded that RAR negative hormones mediate repression ofRAR-mediated transcriptional activity by promoting increased interactionbetween the RAR and NCPs. Further, we have demonstrated that AGN 193109can potentiate the effects of agonists of other nuclear receptors in amanner consistent with the mutual sharing of NCPs between members of thesteroid superfamily of nuclear receptors. As such, ligands can bedesigned and screened to identify compounds having negative hormoneactivity at these non-RAR nuclear receptors.

Our method of RAR negative hormone screening based on the use of CV-1cells co-transfected with the ERE-tk-Luc luciferase reporter plasmid andthe ER-RXR-α and RAR-γ-VP-16 receptor expression plasmids can be adaptedgenerally such that the RAR-γ moiety of the RAR-γ-VP-16 plasmid isconverted to that of peroxisome proliferator-activated receptors (PPAR),vitamin D receptor (VDR), thyroid hormone receptor (T3R) or any othersteroid superfamily nuclear receptor capable of heterodimerizing withRXR. CV-1 cells co-transfected with such plasmids would express highbasal levels of luciferase activity. Ligands capable of binding theligand binding domain of the receptor substituted for the RAR-γ moietycan be easily screened for negative hormone activity by measuring theirability to repress luciferase activity.

For steroid superfamily nuclear receptors that do not heterodimerizewith RXR (e.g., glucocorticoid and estrogen receptors) the same endresult can be achieved using GR-VP-16 or ER-VP-16 receptors and aluciferase reporter plasmid consisting of the appropriate glucocorticoidor estrogen response element fused to a heterologous promoter elementand luciferase or other reporter gene. An essential feature of ageneralized negative hormone screening assay is the inclusion of atleast the ligand binding domain of the particular nuclear receptor forwhich inverse agonists are to be screened and a method for localizingthe nuclear receptor ligand binding domain to the promoter of a reportergene. This could be achieved using the receptors's natural DNA bindingsite, or alternatively by construction of a chimeric receptor having aheterologous DNA binding domain and corresponding use of a reporter genewhich is under control of a DNA regulatory element which is recognizedby the heterologous DNA binding domain. In a preferred embodiment, theplasmid expressing the nuclear receptor for which inverse agonists areto be screened would express this nuclear receptor as a fusion proteincontaining a constitutive activation domain, such as the HSV VP-16activation domain, in order to provide allow high basal activity. Thishigh basal activity would effectively increase assay sensitivity,thereby allowing analysis of nuclear receptor ligands which repressbasal transcriptional activity in the absence of added nuclear receptoragonist.

The following Example illustrates one method that can be used to screenfor compounds having negative hormone activity at the thyroid hormonereceptor.

Example 26 Method of Identifying Thyroid Hormone Receptor NegativeHormones

CV-1 cells are co-transfected with the luciferase reporter plasmidERE-tk-Luc and the plasmids ER-RXR-α and T3R-VP-16. T3R-VP-16 isidentical to the plasmid RAR-γ-VP-16, except the RAR-γ moiety ofRAR-γ-VP-16 has been substituted by the thyroid hormone receptor cDNA.As such, T3R-VP-16 expresses a fusion protein containing the activationdomain of HSV VP-16 in frame with the N-terminus of the thyroid hormonereceptor. Standard transfection and cell culture methods are employedfor this purpose. After transfection, cells are rinsed and fed withgrowth medium containing 10% fetal calf serum which has been extractedwith activated charcoal. Cells are treated with vehicle alone (ethanol),thyroid hormone (10⁻⁹ to 10⁻¹⁰ M), or compound TR-1 (10⁻⁹ to 10⁻⁶ M).TR-1 is a synthetic thyroid hormone receptor ligand which exhibitsstrong affinity for the thyroid hormone receptor in competition bindingstudies, but which does not activate transfected thyroid hormonereceptor in transient cotransfection transactivation assays using athyroid hormone responsive reporter gene and a thyroid hormone receptorexpression plasmid. Further, TR-1 is capable of antagonizing thyroidhormone mediated transactivation and as such is a thyroid receptorantagonist.

Analysis of luciferase activity from CV-1 cell transfected withERE-tk-Luc, ER-RXRα and T3R-VP-16 demonstrates a high basal level ofluciferase reporter activity in vehicle-treated cells. Cells treatedwith thyroid hormone show a slight increase of luciferase activity in adose dependent manner. Cells treated with TR-1 exhibit a dose dependentdecrease in luciferase activity. This indicates that TR-1 exhibitsthyroid receptor inverse agonist activity, presumably due to theincreased interaction of a NCP with the thyroid hormone receptor.

The proliferation rate of human primary retinal pigment epithelium cellsis repressed by treatment with RAR agonists. The therapeutic value ofthis observation has been demonstrated in post-operative use retinoidtherapy after retinal reattachment surgery. We have above demonstratedthe AGN 193109 RAR negative hormone can sensitize primary RPE cells tothe antiproliferative effect of ATRA and 13-cis retinoic acid incoadministration procedures. Further, AGN 193109 was also shown tosensitize RPE cells to the antiproliferative effects of other nuclearreceptor agonists. More specifically, AGN 193109 sensitized RPE cells tothe antiproliferative effects of the glucocorticoid agonist,dexamethasone, and the thyroid hormone agonist 3,3',5-triiodothyronine,T3. This data was consistent with our working model wherein AGN 193109modulated the availability of NCPs that were shared between the membersof the nuclear receptor family. Treatment of RPE cells with the thyroidhormone receptor inverse agonist TR-1 will similarly alter theavailability of shared NCPs such that coadministration with anon-thyroid receptor agonist, such as the RAR agonist 13-cis retinoicacid will lead to an increased antiproliferative effect upon the RPEcultures as compared to 13-cis retinoic acid as a single agenttreatment.

The following Example illustrates one method that can be used to renderprimary RPE cells more sensitive to the antiproliferative activity of anRAR agonist. Notably, this Example further illustrates how the activityof RAR agonists can be potentiated by coadministration with a negativehormone.

Example 27 Sensitizing Primary Retinal Pigment Epithelium Cells to theAntiproliferative Effects of RAR Agonists by Coadministration of theTR-1 Thyroid Hormone Inverse Agonist

Human primary RPE cells are obtained and cultured according to standardmethods. The cultured cells are divided into four groups and treated asfollows. Group 1 receives vehicle alone (ethanol). Group 2 is treatedwith 13-cis retinoic acid at concentrations ranging from 10⁻¹¹ to 10⁻⁶M. Group 3 is treated with the thyroid hormone inverse agonist TR-1 atconcentrations ranging from 10⁻¹¹ to 10⁻¹ M. Group 4 is co-treated with13-cis retinoic acid at concentrations ranging from 10⁻¹¹ to 10⁻⁶ MTR-1. Cells are refed with fresh growth medium and re-treated with theappropriate compound every two days for a total of five days oftreatment. The proliferation rate over the duration of the experiment isquantitated via measurement of the cell number in the cultures using anelectric cell counter.

TR-1 treated cells (Group 3) exhibits rates of cellular proliferationwhich are essentially the same as control (Group 1) cells and there isno effect of this inverse agonist upon the measured growth rate of thecultures. Cells treated with 13-cis retinoic acid (Group 2) exhibit adose dependent decrease in cell number. Comparison of the dose dependentdecrease in cellular proliferation of Group 4 cells (13-cis RA and TR-1coadministration) with that obtained in Group 3 demonstrates the abilityof TR-1 thyroid hormone receptor inverse agonist coadministration tosensitize RPE cultures to the antiproliferative effect of 13-cisretinoic acid as measured by the shift in the dose response curve ofthis RAR agonist to the left in Group 4 as compared to Group 2 cells.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TCAGGTCACCAGGAGGTCAGA21                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AGAAGCTTATGGAAGCAATTATGAGTCAGTTTGCGGGTGACTCTGCAAATACTGCCACTC60                TATAAAAGTTGGGCTCAGAAAGGTGGACCTCGAGGATCCAG101                                  (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 101 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CTGGATCCTCGAGGTCCACCTTTCTGAGCCCAACTTTTATAGAGTGGCAGTATTTGCAGA60                GTCACCCGCAAACTGACTCATAATTGCTTCCATAAGCTTCT101                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTACAAGGTTCACGAGGTTCACGTCTTA28                                                (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TCAGGTCATGACCTGA16                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CCACCCATGGCAAATTCCATGGCA24                                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TCTAGACGGCAGGTCAGGTCCACC24                                                    (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ACGCGTCCGGAAGACCTGGT20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (v) FRAGMENT TYPE:                                                            (vi) ORIGINAL SOURCE:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ATTCTGCAGGTACATGTCCA20                                                        __________________________________________________________________________

What is claimed:
 1. A method of identifying retinoid negative hormones,comprising the following steps:obtaining transfected cells containing arecombinant retinoid receptor and a reporter gene transcriptionallyresponsive to binding of the recombinant retinoid receptor, saidrecombinant retinoid receptor comprising a ligand regulatedtransactivation domain and a dimerization domain; measuring a basallevel of reporter gene expression in said transfected cells whenpropagated in the absence of an added retinoid; treating the transfectedcells with a retinoid compound to be tested for negative hormoneactivity, thereby obtaining treated cells; measuring a lower level ofreporter gene expression in treated cells compared to said basal level,thereby identifying said retinoid compound as a retinoid negativehormone.
 2. The method of claim 1, further comprising the stepsof:coadministering to a collection of cells growing in culture thenegative hormone identified in the second measuring step, and an agonistof a steroid receptor superfamily member, said agonist having apharmacologic activity; and determining whether the coadministerednegative hormone potentiates the pharmacologic activity of the agonist,wherein the negative hormone is identified as a negative hormone havingpotentiating activity if the coadministered negative hormone doespotentiate the pharmacologic activity of the agonist.
 3. The method ofclaim 1 or claim 2, wherein said ligand regulated transactivation domainand said dimerization domain are a retinoid X receptor selected from thegroup consisting of RXR-α, RXR-β and RXR-γ.
 4. The method of claim 1 orclaim 2, wherein the recombinant retinoid receptor is selected from thegroup consisting of RARs and RXRs.
 5. The method of claim 1 or claim 2,wherein the recombinant retinoid receptor is a chimeric retinoidreceptor having a constitutive transcription activator domain.
 6. Themethod of claim 5, wherein the constitutive transcription activatordomain comprises a plurality of amino acids having a net negativecharge.
 7. The method of claim 5, wherein the constitutive transcriptionactivator domain has an amino acid sequence of a viral transcriptionactivator domain.
 8. The method of claim 7, wherein the viraltranscription activator domain is the herpes simplex virus VP-16transcription activator domain.
 9. The method of claim 5, wherein theconstitutive transcription activator domain has a net negative charge,and wherein the recombinant retinoid receptor has deleted therefrom aDNA binding domain.
 10. The method of claim 1 or claim 2, wherein therecombinant retinoid receptor has a DNA binding domain specific for acis-regulatory element other than a retinoic acid responsive element.11. The method of claim 10, wherein the cis-regulatory element otherthan a retinoic acid responsive element is an estrogen responsiveelement.
 12. The method of claim 1 or claim 2, wherein the transfectedcell is propagated in a growth medium substantially depleted ofendogenous retinoids.
 13. The method of claim 12, wherein the growthmedium comprises activated charcoal-extracted serum.
 14. The method ofclaim 1 or claim 2, wherein the reporter gene is the luciferase gene andwherein the measuring steps comprise luminometry.
 15. The method ofclaim 1 or claim 2, wherein the reporter gene is the β-galactosidasegene and wherein the measuring steps comprise a β-galactosidase assay.16. The method of claim 1 or claim 2, wherein the transfected cell is atransfected mammalian cell.
 17. The method of claim 16, wherein thetransfected mammalian cell is a transiently transfected mammalian cell.18. The method of claim 16, wherein the transfected mammalian cell is atransfected Green monkey cell.
 19. The method of claim 16, wherein thetransfected mammalian cell is a transfected human cell.
 20. The methodof claim 1 or claim 2, wherein said ligand regulated transactivationdomain and said dimerization domain are derived from a retinoic acidreceptor selected from the group consisting of RAR-α, RAR-β and RAR-γ.